Polypeptide of protein p140 and DNAs encoding it

ABSTRACT

The present invention is related to a novel protein p140 polypeptide which is a key protein involved in the signal transmission system of insulin; method for preparation of it; DNA encoding the said polypeptide; vector derived the said DNA; host cells transformed the said vector; antibody of the said polypeptide; pharmaceutical composition containing the said peptide or antibody; method for the prevention and/or treatment of diabetes, which is characterized by tyrosine phosphorylation of the said protein p140; agent for the prevention and/or treatment for the currently said the prevention and/or treatment method; agent for the prevention and/or treatment of diabetes, which is characterized by containing a compound which can tyrosine phosphorylation of protein p140, as active ingredient and the screening methods of the said prevention and/or treatment agent. 
     Tyrosine phosphorylation of protein p140 is an essential step in the induction of hypoglycemia by glucose uptake. Method and agent of prevention and/or treatment based on tyrosine phosphorylation of protein p140 in the present invention is not only improve the diabetes-derived hyperglycemic conditions but are also useful for the treatment and/or prevention of diabetes, especially non-insulin dependent diabetes mellitus (NIDDM).

This is a divisional of Application No. 09/192,435 filed Jan. 8, 1998and issued as U.S. Pat. No 6,303,320, which is a divisional ofApplication No. 08/571,785 filed Dec. 13, 1995 and issued as U.S. Pat.No. 5,804,411, which is a divisional of Application No. 08/348,143 filedNov. 23, 1994 and issued as U.S. Pat. No. 5,506,205, the disclosures ofwhich are incorporated herein by reference.

SUMMARY

The present invention is related to a novel protein p140 polypeptidewhich is a key protein involved in the signal transmission system ofinsulin; method for preparation of it; DNA encoding the saidpolypeptide; vector derived the said DNA; host cells transformed thesaid vector; antibody of the said polypeptide; pharmaceuticalcomposition containing the said peptide or antibody; method for theprevention and/or treatment of diabetes, which is characterized bytyrosine phosphorylation of the said protein p140 (to be quotedhenceforth as phosphorylation in the present detailed specification);agent for the prevention and/or treatment for the currently said theprevention and/or treatment method; agent for the prevention and/ortreatment of diabetes, which is characterized by containing a compoundwhich can tyrosine phosphorylate of protein p140, as active ingredientand the screening methods of the said prevention and/or treatment agent.

BACKGROUND OF INVENTION

Diabetes, an abnormal metabolic disease, is induced by a defect in themechanism of glucose metabolism.

Under normal conditions, glucose metabolism occurs as follows:Carbohydrates, consumed in the form of food, are digested to glucose inthe intestines prior to absorption into the circulatory system.Pancreatic β cells respond to an increase in the blood glucose level bysecreting insulin, which in turn stimulates the target peripheraltissues (muscles and liver) to decrease the blood glucose level byenhancing tissue absorption of the blood glucose followed by conversionto glycogen for storage.

Depending on the causative factors, diabetes is classified into twomajor categories; insulin dependent diabetes mellitus (IDDM) andnon-insulin dependent diabetes mellitus (NIDDM). IDDM (Type I diabetes)is a pathological condition where insulin is not secreted orinsufficient even on secretion by pancreatic β cells responding to anincrease in the blood glucose level induced by food consumption. It hasbeen known that destruction of β cells of the pancreatic islets inducesIDDM. The current therapy employs supplementation of insulin fromexogenous sources.

NIDDM (Type II diabetes) is a pathological condition where the feedbackmechanism of peripheral tissues is dysfunctional and is ineffective indecreasing the blood glucose level although normal insulin secretionoccurs within the living system. In the United States of America, NIDDMis said to be a common disease; 5% of the population exceeding 40 yearsof age suffer from NIDDM. Causative factors involved in this diseasehave yet to be elucidated.

RELATED ARTS

Elucidation of the etiology of NIDDM; namely, clarification of theinsulin-induced glucose uptake mechanism in peripheral tissue cells is,however, unclear as current knowledge on information transmissionmechanism of insulin remains limited and unestablished.

Insulin secreted from the pancreatic islets binds with insulin receptorson the cell membrane of peripheral tissue cells. With regards topost-binding information transmission, the phosphorylase cascade andsecond messenger theories are the current topics of research.

Briefly, these two theories can be accounted as follows:

Phosphorylase Cascade Theory:

When insulin binds with the insulin receptor β subunit, the α subunitexisting on the inner cell membrane triggers phosphorylation accompaniedby activation of the tyrosine kinase site within the receptor.Phosphorylation of substrates by the latter enzyme produces threedifferent proteins. One is composed of 1,235 amino acids and has amolecular weight of 185 kD corresponding to the insulin receptorsubstrate-1 (IRS-1). On tyrosine phosphorylation of IRS-1, thephosphorylase for phosphatidylinositol, PI1-kinase, binds against andactivates the complex. Post-binding events related to informationtransmission that concerns localization of glucose transporter withinthe membrane and membrane ruffling have yet to be established. Otherthan IRS-1, the existence of two protein substrates (Shc and PTP-1C) hasbeen confirmed. However, the follow-up mechanism(s) has not beencompletely accounted for.

Second Messenger Theory:

When insulin binds against the insulin receptor, phospholipase C isspecifically activated to degrade phosphatidylinositol glycan (PIG) toproduce inositolglycan (IG) and diacylglycerol (DAG) by hydrolysis.Although IG has been reported to display various insulin-like effects,the typical glucose uptake effect has yet to be demonstrated.

However, when protein kinase C is activated by DAG, localization ofprotein kinase C within the cell membrane has been known to be promoted.This implicates that DAG sequentially phosphorylates inner membraneproteins to finally trigger the glucose uptake. However, thisimplication remains hitherto unclear.

Although the two different schools of thought have hitherto prevailed,initial stages of the post-binding events related to informationtransmission can only be explained in part by either theory.

According to Copper et al. in 1988 the hormone, amylin, is released fromb pancreatic cells that similar to those that secret insulin whenhyperglycemia prevails. Based on their findings that amylin inhibitedthe action of insulin, they revealed that the hormone might be used asan insulin antagonist. A follow-up report in 1991 indicates that theexcessive use of amylin in transgenic mice induces NIDDM. However, therelationship of amylin with insulin information transmission remainshitherto unexplored.

MEANS TO SOLVE THE PROBLEMS

The inventors of the present invention focus on the insulin antagonisticproperties of amylin. With persistent research activities conducted onthe effects of amylin on the insulin information transmission system,the inventors first identified the inhibition site of amylin inregulating the insulin information transmission system and discoveredthe key proteins, phosphorylated protein 140 and 70 (pp140 and pp70),related to this phenomenon. The present invention reveals clearly thestructures of said proteins (DNA base sequences and amino sequences) andelucidation of their functions to totally complement the hithertodeficiently explained insulin information transmission phenomenon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an effects of vitamin K₅ (VK₅) on blood glucose contents instreptozotocin (STZ)-induced diabetic rats.

FIG. 2 shows an effects of vitamin K₅ (VK₅) on neutral fat contents inblood of streptozotocin (STZ)-induced diabetic rats

FIG. 3 shows an effects of vitamin K₅ (VK₅) on blood cholesterolcontents in streptozotocin (STZ)-induced diabetic rats

FIG. 4 shows a hydrophobicity profile for the polypeptide of proteinp140 in the present invention

FIG. 5 shows the pUCSRαML2 vector.

DISCLOSE OF THE INVENTION

The present invention related to homologues and fragment sequences ofthe genuine amino acid sequence of the said protein p140 constructedfrom SEQ ID No.1 as shown. In addition, DNAs encoding the relatedpolypeptides of the said homologues and fragment sequences are alsoencompassed in present invention. Expressed on a more concrete aspect,the said DNAs are those either encoding and/or possessing fragmentsselectively hybridizing base sequences illustrated in SEQ ID No.2 and 3.

Furthermore, in the present invention, method for the prevention and/ortreatment of diabetes, which is characterized by tyrosinephosphorylation of the said protein p140; agent for the preventionand/or treatment for the currently said the prevention and/or treatmentmethod; agent for the prevention and/or treatment of diabetes, which ischaracterized by containing a compound which can tyrosinephosphorylation of protein p140, as active ingredient and the screeningmethods of the said prevention and/or treatment agent.

The present invention specifically include:

(1) polypeptides constructed by amino sequence(s) illustrated in SEQ IDNo.1.

(2) DNAs encoding polypeptides described in (1).

(3) DNAs possessing base sequences illustrated in SEQ ID No.2.

(4) DNAs possessing base sequences illustrated in SEQ ID No.3.

(5) Method for the prevention and/or treatment of diabetes, which ischaracterized by tyrosine phosphorylation of protein p140

(6) Agent for the prevention and/or treatment of diabetes, which ischaracterized by tyrosine phosphorylation of protein p140

(7) Agent for the prevention and/or treatment of diabetes, which ischaracterized by containing a compound which can tyrosinephosphorylation of protein p140, as active ingredient , and

(8) Method for the screening of the agent for the prevention and/ortreatment of diabetes, which is characterized by using protein p140.

On administering amylin (0.1 mg/kg, i.p., t.i.d.) to healthy rats for 7days, dramatic decreases in both incidences of insulin receptorpopulation and secreted insulin quantity. These observations wereaccompanied by decreases in both incidences, glucose transporter 4 (Glut4) quantity and synthesized glycogen content (less than 50% decreasecompared to that of control group) with 1.7-fold increase in the bloodglucose content. Furthermore, in experiments using L6 cells (ATCC strainNo., CRL-1458) of rat skeletal muscle myoblasts, a decreased glucoseuptake in the cells was observed with amylin administration.

Next, changes in the insulin-induced tyrosine phosphorylation cascade inskeletal muscle myoblasts treated with amylin were investigated by usingthe anti-phosphotyrosine antibody with the western blot method. As such,when L6 cells were incubated with insulin in the experiments, tyrosinephosphorylation was enhanced. However, pretreatment with amylin undersimilar conditions confirmed the presence of two different proteins thatinhibited the phosphorylation. These proteins are henceforth termed aspp140 and pp70 according to their respective molecular weights.Furthermore, the precursors of these said proteins prior tophosphorylation are, however, henceforth designated as p140 and p70respectively.

The inventors prepared, isolated and purified the pp140 and pp70 beforedetermining their partial amino acid sequences. On comparingsimilarities of the said amino acid sequences with previously documentedsequences of polypeptides in Swiss Plot Release 2.0, pp70 coincides withthe previous known glucose-regulated protein 70. However, the resultspostulate pp140 as a totally unknown novel protein. As such, inventorsof the present invention isolated mRNA of p140 from the rat skeletalmuscle myoblasts and constructed the cDNA using the isolated mRNA ofp140 before determining the whole base sequence and complete amino acidsequence of the said protein. The results therefore complement thepresent invention by revealing successfully a completely novelpolypeptide and the total DNA chain encoding this polypeptide.

From the above findings, it is understood that amylin may inhibitphosphorylation of p140 and p70 into pp140 and pp70 respectively. Incontrast, when amylin is considered to suppress the process from insulinreceptor binding to glucose uptake, it suggests that phosphorylation ofp140 and p70 to yield pp140 and pp70 may play an important role in theglucose uptake mechanism of cells.

The inventors of the present invention attempted to elucidate themechanism(s) of action of p140 and p70 accordingly.

When rat skeletal muscle myoblasts (rat L6 cells) were incubated ininsulin-supplemented cultures, incidence of a pp140 band on day 3 withpp140 production on day 9 were persistently observed. At about thesimilar interval (day 3), incidence of Glut 4 was similarly observedwith gradual increases in rat L6 cell division. Furthermore,polynucleation of rat skeletal muscle myoblasts was observed on day 7 inthe similar culture system with subsequent division to form the musclecells. In the case of pp140, the cells appeared on day 7 and persistedto register production of the protein until day 14.

However, on examining localization of pp140 within the cells, the saidprotein was found within the microsome membrane (MM) of cytoplasm in thecell at post-culture 10 min when insulin was added to non-serum treatedL6 cells. The pp140 disappeared thereafter. In addition, pp140 was firstobserved in the cell permeable membrane (PM) at post-culture 1˜hr. Fromthese findings, pp140 is postulated to have synthesized in cellcytoplasm immediately after insulin treatment ensued with transfer ofthis protein to permeable membrane (PM) 1˜2 hr thereafter. Furthermore,when pp70 localization in L6 cells was investigated with a similarexperimental approach, pp70 was first located in the MM immediatelyafter initiating the culture, registered a peak phosphorylated quantityat post-culture 10 min and gradually approached non-detectable values atpost-culture 3 hr. Moreover, pp70 was also located within the nucleusimmediately after initiating the culture, and the protein contentgradually increased to register a peak value at post-culture 3 hr. Fromthe above protein localization patterns, pp70 exists in MM in theabsence of insulin and this protein is mobilized to the nucleus fractionwithin 3 hr after insulin treatment.

Based on the above results, pp140 information transmission mechanism maybe postulated as follows. In short, when insulin binds to the receptor,the latter is activated by auto-phosphorylation. The information is thensubjected to undergo various steps of activation via phosphorylation ofprotein phosphorylases to subsequently phosphorylate p140 to pp140. Theactivated pp140 localizes on permeable membrane (PM) surface before p70is phosphorylated after undergoing various protein phosphorylationprocesses simultaneously. The phosphorylated pp70 is activated thenmobilized to within the nucleus to subsequently trigger biologicalactivities in the Glut 4 expression within the nucleus. Based on thisinformation, Glut 4 produced within the cytoplasm is hence mobilized tolocalize on the permeable membrane (PM) surface to eventually triggerglucose uptake.

The above information transmission mechanism warrants follow-upexperiments to righteously establish concrete evidence of thephenomenon. In any case, it can now be concluded that activation of p140is an essential step required to induce glucose uptake in cells andsubsequent hypoglycemia in the circulatory system.

As such, the present invention is related to method for the preventionand/or treatment of diabetes, especially non-insulin dependent diabetesmellitus (NIDDM), which is characterized by tyrosine phosphorylation ofprotein p140.

Moreover, the present invention is related to agent for the preventionand/or treatment of diabetes, especially non-insulin dependent diabetesmellitus (NIDDM), which is characterized by tyrosine phosphorylation ofprotein p140.

In the present invention, method and agent for the prevention and/ortreatment of diabetes, which is characterized by tyrosinephosphorylation of protein p140, includes all or whole of the saidmethod and agent for the prevention and/or treatment of diabetes basedon the major mechanism of action involving tyrosine phosphorylation ofprotein p140.

In addition, cells that tyrosine phosphorylate protein p140 are not onlyconfined to skeletal muscle myoblasts (rat L6 cells), but also includeall other cells that positively elicits the said phosphorylation. All inall, cells that have been confirmed to display the said phosphorylationinclude rat FaO hepatocytes, human A673 muscle cells and HepG2hepatocytes.

Organs other muscles and liver such as the heart, brain, spleen, lungs,kidneys, testes, placenta and pancreas have repeatedly displayedincidences of p140 mRNA of the present invention. Without being confinedmerely to muscles and liver, the effects of tyrosine phosphorylation maytherefore radiate extensively throughout the living system. From thisfinding, the said mechanism of action of the present invention is hencenot limited to muscle and liver cells, but involves the cardiac,encephalic, splenic, pulmonary, renal testical, placental and pancreaticcells as well.

When the polypeptide of the present invention was compared with aminoacid sequences of previously known polypeptides recorded with the SwissProt Release 2.0, candidates with a complete whole sequence similar tothat of the polypeptide was not identified. Furthermore, no a singlecDNA of the complete whole polypeptide of the present invention encodingthe previously documented nucleotide sequences recorded in the GenBankRelease 70 was located. The said peptide of the present invention ishence confirmed to be a completely novel protein.

Additionally, epiterial cell kinase (Eck) and approximately 40% identitywere recognized when the results were compared with amino acid sequencesof polypeptides previously documented in the Swiss Prot Release 2.0. Assuch, a novel protein of the present invention was postulated to belongto the Eck family.

In the present invention, a polypeptide of Seq. ID No. 1 insubstantially purified form will generally comprise the polypeptide in aproduction in which more than 90%, e.g. 95%, 98% or 99% of thepolypeptide in the production is that of the Seq. ID No. 1.

A polypeptide homologue of the Seq. ID No. 1 will be generally at least70%, preferably at least 80 or 90% and more preferably at least 95%homologous to the polypeptide of Seq. ID No. 1 over a region of at least20, preferably at least 30, for instance 40, 60 or 100 more contiguousamino acids. Such polypeptide homologues will be referred to below as apolypeptide according to the invention.

Generally, fragments of Seq. ID No. 1 or its homologues will be at least10, preferably at least 15, for example 20, 25, 30, 40, 50 or 60 aminoacids in length, and are also encompassed by the term “a polypeptideaccording to the invention” as used herein.

A DNA capable of selectively hybridizing to the DNA of Seq. ID No. 2 or3 will be generally at least 70%, preferably at least 80 or 90% and morepreferably at least 95% homologous to the DNA of Seq. ID No. 2 or 3 overa region of at least 20, preferably at least 30, for instance 40, 60 or100 or more contiguous nucleotides. Such DNA will be encompassed by theterm “DNA according to the invention”.

Fragments of the DNA of Seq. ID No. 2 or 3 will be at least 10,preferably at least 15, for example 20, 25, 30 or 40 nucleotides inlength, and are also encompassed by the term “DNA according to theinvention” as used herein.

A further embodiment of the invention provides replication andexpression vectors comprising DNA according to the invention. Thevectors may be, for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said DNA and optionally a regulator of the promoter. The vectormay contain one or more selectable marker genes, for example aanpicillin resistance gene. The vector may be used in vitro, for exampleof the production of RNA corresponding to the DNA, or used to transfector transform a host cell.

A further embodiment of the invention provides host cells transformed ortransfected with the vectors for the replication and expression of DNAaccording to the invention, including the DNA SEQ. ID No. 2 or 3 or theopen reading frame thereof. The cells will be chosen to be compatiblewith the vector and may for example be bacterial, yeast, insect ormammalian.

A further embodiment of the invention provides a method of producing apolypeptide which comprises culturing host cells of the presentinvention under conditions effective to express a polypeptide of theinvention. Preferably, in addition, such a method is carried out underconditions in which the polypeptide of the invention is expressed andthen produced from the host cells.

DNA according to the invention may also be inserted into the vectorsdescribed above in an antisense orientation in order to proved for theproduction of antisense RNA. Antisense RNA may also be produced bysynthetic means. Such antisense RNA may be used in a method ofcontrolling the levels of a polypeptide of the invention in a cell.

The invention also provides monoclonal or polyclonal antibodies to apolypeptide according to the invention. The invention further provides aprocess for the production of monoclonal or polyclonal antibodies to thepolypeptides of the invention. Monoclonal antibodies may be prepared byconventional hybridoma technology using a polypeptide of the inventionor a fragment thereof, as an immunogen. Polyclonal antibodies may alsobe prepared by conventional means which comprise inoculating a hostanimal, for example a rat or a rabbit, with a polypeptide of theinvention and recovering immune serum.

The present invention also provides pharmaceutical compositionscontaining a polypeptide of the invention, or an antibody thereof, inassociation with a pharmaceutically acceptable diluent and/or carrier.

The polypeptide of the present invention includes that which a part oftheir amino acid sequence is lacking (e.g., a polypeptide comprised ofthe only essential sequence for revealing a biological activity in anamino acid sequence shown in SEQ ID No.1), that which a part of theiramino acid sequence is replaced by other amino acids (e.g., thosereplaced by an amino acid having a similar property) and that whichother amino acids are added or inserted into a part of their amino acidsequence, as well as those having the amino acid sequence shown in SEQID NO. 1.

As known well, there are one to six kinds of codon as that encoding oneamino acid (for example, one kind of codon for Met, and six kinds ofcodon for Leu) are known. Accordingly, the nucleotide sequence of DNAcan be changed in order to encode the polypeptide having the same aminoacid sequence.

The DNA of the present invention, specified in (2) includes a group ofevery nucleotide sequences encoding polypeptides (1) shown in SEQ ID NO.1 . There is a probability of improving a yield of production of apolypeptide by changing a nucleotide sequence.

The DNA specified in (3) is the embodiment of DNA shown in (2), and issequence in the natural form.

The DNA shown in (4) indicates the sequence of the DNA specified in (3)with a non-translational region.

The DNA having a nucleotide sequence shown in SEQ ID NO. 3 may beprepared according to the following methods, that is:

(i) by isolating mRNA from a cell line which produces the polypeptide ofthe present invention (e.g., rat skeletal muscle myoblasts L6 cell),

(ii) by preparing first strand (single stranded DNA) from mRNA thusobtained, followed by preparing second strand (double stranded DNA)(synthesis of cDNA),

(iii) by inserting cDNA thus obtained into a proper plasmid vector,

(iv) by transforming host cells with the recombinant DNA thus obtained(preparation of cDNA library),

(v) by random-cloning on a large scale from cDNA library thus obtained,followed by sequencing average 300 bases from 5′ end of each clone, and

(vi) by sequencing complete length of a clone which has a novel basesequence.

Explained in detail, step (i) may be carried out in accordance with themethod of Okayama, H. et al. (described in Methods in Enzymology, 154,3, (1987)) using L6 cells of a rat skeletal muscle myoblasts which islogarithmic growth phase. Examples of the cells which produce thepolypeptide of the present invention is rat or human of muscle, liver,heart, brain, spleen, lungs, kidneys, testes, placenta or pancreas, andis preferably rat skeletal muscle myoblasts L6 cell (ATCC strain No.,CRL-1458), rat liver FaO cell, human muscle A673 cell or human liverHepG2 cell. Steps (ii), (iii) and (iv) are a series of steps forpreparing cDNA library, and may be carried out in accordance with themethod of Gubler & Hoffman (Gene, vol. 25, pp. 263, 1983) with a slightmodification. As examples of the plasmid vector used in the step (iii),many vectors functioning in an E. coli strain (e.g., pBR 322) and in aBacillus subtilis (e.g., pUB 110) are known, and pGEM-3Zf(+) (3,199 bp,manufactured by Promega Corp.) which functions in an E. Coli, may bepreferably used. As examples of host used in the step (iv), many cellsare already known. Any cells may be used, and DH5 competent cell whichhas been prepared in accordance with the method described in Gene, vol.96, pp. 23, 1990, may be preferably used. The cloning in the step (v)may be carried out by methods known per se and the sequencing may becarried out in accordance with the method of Maxam-Gilbert or thedideoxy termination method. The step (vi).may be carried out inaccordance with the method described in Molecular Cloning (written bySambrook, J., Fritsch, E. F. and Maniatis, T., published by Cold SpringHarbor Laboratory Press in 1989).

As the following step, it is necessary to examine whether or not the DNAthus obtained codes right a produce,protein. The examination requires:

(I) the conversion of the DNA sequence into the amino acid sequence in apossible frame,

(II) the confirmation that the DNA thus obtained covers complete oralmost complete length of intact mRNA. These confirmation may be carriedout after the step (vi) hereinbefore described, and effectively betweenthe step (v) and the step (vi).

The step (II) may be carried out by Northern analysis.

Once the nucleotide sequences shown in SEQ ID NOs. 2 and 3 aredetermined, DNA of the present invention may be obtained by chemicalsynthesis, by PCR method or by hybridization making use of a fragment ofDNA of the present invention, as a probe. Furthermore, DNA of thepresent invention may be obtained in a desired amount by transformingwith a vector DNA inserted a DNA of the present invention into a properhost, followed by culturing the transformant.

The polypeptides of the present invention (shown in SEQ ID NO. 1) may beprepared by:

(1) isolating and purifying from an organism or a cultured cell,

(2) chemically synthesizing, or

(3) using a skill of biotechnology, preferably, by the method describedin (3).

Examples of expression system when preparing a polypeptide by using askill of biotechnology is, for example, the expression system ofbacteria, yeast, insect cell and mammalian cell.

For example, the expression in E. coli may be carried out by adding theinitiation codon (ATG) to 5′ end of a DNA encoding a nucleotide sequenceshown in SEQ ID NO. 3, connecting the DNA thus obtained to thedownstream of a proper promoter (e.g., trp promoter, lac promoter,λP_(L) promoter, T7 promoter etc.), and then inserting it into a vector(e.g., pBR322, pUC18, pUC19 etc.) which functions in an E. coli strainto prepare an expression vector. Then, an E. coli strain (e.g., E. coliDH1 strain, E. coli JM109 strain, E. coli HB101 strain, etc.) which istransformed with the expression vector thus obtained may be cultured ina proper medium to obtain the desired polypeptide. When a signal peptideof bacteria (e.g., signal peptide of pel B) is utilized, the desiredpolypeptide may be also secreted in periplasm. Furthermore, a fusionprotein with other polypeptide may be also produced easily.

Furthermore, the expression in a mammalian cell may be carried out, forexample, by inserting the DNA shown in SEQ ID NO. 3 into the downstreamof a proper promoter (e.g., SV40 promoter, LTR promoter, metallothioneinpromoter etc.) in a proper vector (e.g., retrovirus vector, papillomavirus vector, vaccinia virus vector, SV40 vector, etc.) to obtain anexpression vector, and transforming a proper mammalian cell (e.g.,monkey COS-7 cell, Chinese hamster CHO cell, mouse L cell etc.) with theexpression vector thus obtained, and then culturing the transformant ina proper medium to get a desired polypeptide in the culture medium. Thepolypeptide thus obtained may be isolated and purified by conventionalbiochemical methods.

The protein of the present invention includes the reaction products ofphosphorylated and/or sugar-chained protein. In short, the presentinvention contains p140-bound polysaccharide chains and tyrosinephosphorylated p140 (pp140) found in p140 polypeptides.

EFFECTS OF INVENTION

The protein p140 is postulated to possess the above-mentioned mechanismof action. The protein p140 polypeptide of the present invention cantherefore not only improve the hyperglycemic conditions when used alone,but can also be useful in prevention and/or treatment for diabetes,especially non-insulin dependent diabetes mellitus (NIDDM).

Further, polyclonal or monoclonal antibody against the protein p140polypeptide of the present invention can be used in the determination ofthe amount of the said polypeptide in organism, and thereby, may beutilized for the purpose of investigating the relationship between thesaid polypeptide and diseases, or for the purpose of diagnosingdiseases, and the like. Polyclonal and monoclonal antibody thereof maybe prepared by conventional methods by using the said polypeptide or thefragment thereof as an antigen.

The DNA of the present invention may be utilized as an important andessential template in preparing the polypeptide of the present inventionwhich is expected to possess various use or for diagnosis of and in thetreatment of gene diseases (the treatment of gene defect disease and thetreatment by inhibiting expression of the polypeptide by antisense DNA(RNA), and the like). Further, genomic DNA may be isolated by using theDNA of the present invention as a probe. Similarly, it is possible toisolate genes having high homology to the DNA of the present inventionin human or those of other species.

Furthermore, the present invention is related to agent for theprevention and/or treatment of diabetes characterized by containing acompound which can tyrosine phosphorylation of protein p140, as activeingredient.

All in all, tyrosine phosphorylated protein p140 products include notonly currently confirmed substances that possess the said activities butalso all those substances that will be confirmed to possess the saidactivities henceforth. At present, it is confirmed that the compoundshave activity of tyrosine phosphorylation, for example,

(1) the benzene or naphthalene derivatives of the formula (I)

wherein R¹ of n species each, independently, is hydrogen atom C1-4alkyl, hydroxy, amino or COOR² (in which R² is hydrogen atom or C1-4alkyl), n is 1-3 and non-toxic salts thereof and non-toxic acid additionsalts thereof,

(2) the benzoquinone or naphthoquinone derivatives of the formula (II)

wherein R³ of m species each, independently, is hydrogen atom, C1-12alkyl, C1-4 alkoxy, C1-4 alkylthio, hydroxy, halogen, phenyl or phenylsubstituted by halogen, m is 1-4,

(3) the rhodanine or thazolidine derivatives of the formula (III)

wherein X is oxygen or sulfur atom, R⁴ and R⁵ each, independently, ishydrogen atom, phenyl or phenyl substituted by C1-4 alkyl, C1-8 alkoxy,halogen atom or nitro, or R⁴ and R⁵, taken together, representbenzylidene, benzylidene substituted by C1-4 alkyl, C1-8 alkoxy, halogenatom or nitro or β-methylcinnamilidene, R⁶ is hydrogen atom C1-4 alkyl,and non-toxic salts thereof and non-toxic acid-addition salts thereof.

More concretely, the compounds of the formula (I) include4-amino-2-hydroxybenzoic acid, 4-amino-i-naphthol, 4-amino-2-naphthol,1-aminonaphthalene, 1,4-dihydroxynaphthalene, 4-amino-2-methyl-1-naphthol (abbreviated as vitamin K₅ hereinafter), 1,4-dihydroxy-2-naphthenic acid, etc.

The compounds of the formula (II) include 2-methyl-1,4-benzoquinone,2,6-di-tert-butyl-1,4-benzoquinone, 2,6-dibromo-1,4-benzoquinone,2,3,4,5-tetrafluoro-1 ,4-benzoquinone, 1,4-naphthoquinone,2-methyl-1,4-naphthoquinone (abbreviated as vitamin K3 hereinafter),2-hydroxy-3-methyl-1,4-naphthoquinone,2-(3,7-dimethyloctyl)-3-hydroxy-1,4-naphthoqunone,2-methoxy-3-methyl-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone,3-(4-chlorophenyl)-2-hydroxy-1 ,4-naphthoquinone, 2-propylthio- 1,4-naphthoquinone, etc.

The compounds of the formula (Ill) include 5-phenylrhodanine,5-phenyl-1,3-thiazodidine-2,4-dione, 5-benzylidenerhodanine,5-benzylidene-1,3-thiazodidine-2,4-dione, 5,5-diphenylrhodanine,5,5-diphenyl- 1 ,3-thiazodidine-2,4-dione,5-(4-isoamyloxybenzylidene)rhodanine,5-(4-isoamyloxybenzylidene)-1,3-thiazodidine-2,4-diene,5-(β-methylcinnamylidene)rhodanine-3-acetic acid, etc., and non-toxicsalts thereof and non-toxic acid addition salts thereof.

In the present invention, the appropriate non-toxic salts, for example,are salts of alkali metal (e.g., potassium, sodium etc.), salts ofalkaline earth metal (e.g., calcium, magnesium etc.), ammonium salts,salts of pharmaceutically-acceptable organic amine (e.g.,tetramethylammonium, triethylamine, methylamine, dimethylamine,cyclopentylamine, benzylamine, phenethylamine, piperidine,monoethanolamine, diethanolamine, tris(hydroxymethyl)amine, lysine,arginine, N-methyl-D-glucamine etc.).

In the present invention, the appropriate acid addition salts includethe salts with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid and nitric acid, and the salts withorganic acids such as acetic acid, trifluoroacetic acid, lactic acid,tartaric acid, oxalic acid, fumaric acid, maleic acid, benzenesulfonicacid, toluenesulfonic acid, isethionic acid, glucuronic acid andgluconic acid.

The compound of the formulae (I), (II) and (III) are well known per se ,or used as other starting materials are may be easily prepared bymethods known per se.

As the substances used in the present invention are subjected totyrosine phosphorylation, these agents not only improve thediabetes-derived hyperglycemic conditions but are also useful for thetreatment and/or prevention of diabetes, especially non-insulindependent diabetes mellitus (NIDDM).

It was confirmed that the toxicity of the various active ingredient andsalts thereof of the present invention is very low. Therefore, it may beconsidered that the various active ingredient and acid-addition saltsthereof of the present invention are safe and suitable forpharmaceutical use.

For the purpose above described, the polypeptide, each active ingredientand acid addition salts thereof of the present invention, may benormally administered systemically or partially, usually by oral orparenteral administration.

The doses to be administered are determined depending upon e.g., age,body weight, symptom, the desired therapeutic effect, the route ofadministration, and the duration of the treatment. In the human adult,the doses per person per dose are generally between 10 μg and 1000 mg,by oral administration, up to several times per day, and between 10 μgand 100 mg, by parenteral administration up to several times per day, orby continuous intravenous administration between 1 and 24 hrs. per day.

As mentioned above, the doses to be used depend upon various conditions.Therefore, there are cases in which doses lower than or greater than theranges specified above may be used.

When administration of the compounds of the present invention, it isused as solid compositions, liquid compositions or other compositionsfor oral administration, as injections, liniments or suppositories forparenteral administration.

Solid compositions for oral administration include compressed tablets,pills, capsules, dispersible powders, and granules. Capsules includehard capsules and soft capsules.

In such compositions, one or more of the active compound(s) is or areadmixed with at least one inert diluent (such as lactose, mannitol,glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch,polyvinylpyrrolidone, magnesium metasilicate aluminate). Thecompositions may also comprise, as is normal practice, additionalsubstances other than inert diluents: e.g. lubricating agents (such asmagnesium stearate), disintegrating agents (such as cellulose calciumglycolate), stabilizing agents (such as lactose), and agents to assistdissolution (such as glutamic acid, asparaginic acid).

The tablets or pills may, if desired, be coated with a film of gastricor enteric material (such as sugar, gelatin, hydroxypropyl cellulose orhydroxypropylmethyl cellulose phthalate), or be coated with more thantwo films. Coating may include containment within capsules of absorbablematerials such as gelatin.

Liquid compositions for oral administration includepharmaceutically-acceptable solutions, emulsions, suspensions, syrupsand elixirs. In such compositions, one or more of the active compound(s)is or are contained in inert diluent(s) commonly used in the art (suchas purified water, ethanol). Besides inert diluents, such compositionsmay also comprise adjuvants (such as wetting agents, suspending agents),sweetening agents, flavouring agents, perfuming agents, and preservingagents.

Other compositions for oral administration include spray compositionswhich may be prepared by known methods and which comprise one or more ofthe active compound(s). Spray compositions may comprise additionalsubstances other than inert diluents: e.g. stabilizing agents (such assodium sulfate), isotonic buffer (such as sodium chloride, sodiumcitrate, citric acid). For preparation of such spray compositions, forexample, the method described in the U.S. Pat. No. 2,868,691 or3,095,355 may be used.

Injections for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. In such compositions,one more active compound(s) is or are admixed with at least one inertaqueous diluent(s) (such as distilled water for injection, physiologicalsalt solution) or inert non-aqueous diluent(s) (such as propyleneglycol, polyethylene glycol, olive oil, ethanol, POLYSORBATE 80(registered trade mark)).

Injections may comprise additional materials other than inert diluents:e.g. preserving agents, wetting agents, emulsifying agents, dispersingagents, stabilizing agent (such as lactose), and agents to assistdissolution (such as glutamic acid, asparaginic acid).

They may be sterilized for example, by filtration through abacteria-retaining filter, by incorporation of sterilizing agents in thecompositions or by irradiation. They may also be manufactured in theform of sterile solid compositions, for example, by freeze-drying, andwhich may be dissolved in sterile water or some other sterile diluent(s)for injection immediately before used.

Other compositions for parenteral administration include liquids forexternal use, and endermic liniments, ointment, suppositories andpessaries which comprise one or more of the active compound(s) and maybe prepared by methods known per se.

EXAMPLE

The following examples are illustrated, but not limit, the presentinvention.

Example 1 Purification Method of pp140

By employing cell trays (225 cm²), rat L6 cells were incubated at 37° C.for 7˜10 days in 5% CO₂ atmosphere. Culture media were replaced at 3-dayintervals with the Dulbecco's modified Eagle's medium (containing 10%bovine fetal serum (BFS)). Two hours after treating the muscle cellsdeveloped from skeletal muscle myoblasts with serum-free medium, 500 μMvanadic acid (vanadate) was added to the culture and allowed to incubatefurther for 10 min. Cells were then suspended in Tris buffer (400 μMvanadate with protease inhibitor), lysed and centrifuged prior toisolating the supernatant.

The supernatant was adjusted with octa (ethylene glycol) ether (C₁₂E₈)to a final concentration of 0.1% before filtration through a milliporemembrane. Protein G sepharose gel bound with anti-phosphotyrosineantibodies was filled with the filtered sample. The tyrosinephosphorylated protein (pp140) adsorbed to the gel. After rinsing thecolumn with 25 mM Tris buffer, pp140 was eluted with 10 mMphenylphosphate. The eluate was concentrated with Centricon 30 prior toprecipitating pp140 by the acetone precipitation method.

Example 2 Tyrosine Phosphorylation of p140 in Various Tissues

Using the Dubecco's modified Eagle's medium (containing 10% BFS),various cells (1×10₅ cells/dish) were incubated at 37° C. under 5% CO₂atmosphere for 5˜8 days. The cells were skeletal muscle cellsdifferentiated from skeletal muscle myoblasts. The differentiated cellspreviously treated in serum-free Dulbecco's modified Eagle's medium for4 hr was incubated with and without amylin (100 pM) before furtherincubation for 24 hr. Cultures treated with insulin (100 nM) thereafterwere incubated for a fixed interval (10 or 60 min).

After the cultures were rinsed with ice-cold phosphate buffer, cellswere lysed with phosphate buffer containing 0.5% octa (ethylene glycol)ether (C₁₂E₈). The pp140 was recovered by sepharobeads bound withphosphothyrosine antibody (Transformation Corp.) prior to elution anddetection with phenyl phosphate and western blotting method,respectively. The band content of pp140 was determined by a densitometerusing purified pp140 as the standard. The results are illustrated inTable 1.

TABLE 1 Effects of p140 tyrosine phosphorylation on various tissues rathuman L6 FaO A 678 HepG2 control 300 100 300 250 insulin 10 min 24002000 2500 2100 insulin 60 min 1000 1400 1900 1200 insulin added amylin10 min 180 300 350 300 insulin added amylin 60 min 200 100 1000 300

In the Table 1, cultures were treated with amylin (100 pM) 24 hr beforeinsulin (100 nM) was added.

OBSERVATION

Incidence of pp140, observed when rat L6 cells were incubated withinsulin within 10 min, was antagonized by amylin treatment. Moreover,this phenomenon was similarly confirmed in rat hepatocytes, FaO cells.Furthermore, this phenomenon is not merely confined to rats. In humanmuscle cells (A673 cells) and hepatocytes (HepG2 cells), the phenomenonhas been similarly confirmed. It is postulated that amylin suppresses acertain stage or processes before p140 phosphorylation is triggered bythe phosphorylation signal of insulin.

Example 3 Effects of Various Test Compounds on p140 Phosphorylation

Rat L6 cells (1×10⁵ cells/dish) were incubated in the Dulbecco'smodified Eagle's medium (containing 10% BFS) at 37° C. under 5% CO₂atmosphere for 8 days. The cells used were skeletal musclemyoblast-differentiated muscle cells. After treating the differentiatedskeletal muscle cells in serum-free Dulbecco's modified Eagle's mediafor 4 hr, various test compounds (10 mM; except insulin,1 mM) were addedbefore the cultures were further incubated for a fixed interval.

After the cultures were rinsed with ice-cold phosphate buffer, cellswere lysed with phosphate buffer containing 0.5% octa (ethylene glycol)ether (C₁₂E₈). The pp140 was recovered by cephalobeads bound withphosphotyrosine antibody (Transformation Corp.) prior to elution anddetection with phenyl phosphate and western blotting method,respectively. The band content of pp140 was determined by a densitometerusing purified pp140 as the standard. The results are illustrated inTable 2.

TABLE 2 Effects of p140 tyrosine phosphorylation on various testcompounds Amount of tyrosine phosphorylated p140 (copy/cell) Compound 03 10 60 (min) Vitamin K₃ 350 3650 1800 750 Vitamin K₅ 400 3850 2850 16005-phenylrhodanine 350 1600 1250 650 5-benzylidenerhodanine 400 2650 19001350 5-(4-isoamyloxybenzylidene) 400 3200 2250 1600 rhodanine Insulin350 1850 2600 1650 (positive control)

Example 4 Enhancement Activity on Glucose Uptake

Rat L6 cells (1×10⁵ cells/dish) were incubated in Dulbecco's modifiedEagle's medium (containing 10% BFS) at 37° C. under 5% C0₂ atmospherefor 8 days. The cells used were skeletal muscle myoblast-differentiatedskeletal muscle cells. After treating the differentiated skeletal musclecells in serum-free Dulbecco's modified Eagle's medium for 2 hr, varioustest compounds (10 mM; except insulin,l mM) were added before thecultures were further incubated for a fixed interval of 2 hr. Culturesthereafter treated with Crebs-Ringer phosphate buffer (pH: 7.4) for 20min were further incubated with 5 mM ³H-2-deoxyglucose (0.05 mCi/ml). Atthe initial 3 min after incubation, the uptake radioactivity content incells was determined with a liquid syntillation counter. The results areillustrated in Table 3.

TABLE 3 Enhancement activity on glucose uptake Activity on glucoseuptake Compound (pmol/mg protein/min) Control 22.6 Vitamin K₃ 60.9Vitamin K₅ 67.5 5-phenylrhodanine 76.8 5-benzylidenerhodanine 84.25-(4-isoamyloxybenzylidene) 98.4 rhodanine Insulin 106.8 (positivecontrol)

Observation

All compounds that promoted p140 phosphorylation were confirmed toactivate glucose uptake activities (Table 2 and 3).

Example 5 Effects of Vitamin K₅ on Diabetes

The diabetes model using streptozotocin (STZ) was established in maleWistar rats (STZ rats). After administering various intraperitoneal(i.p.) daily doses of vitamin K₅ for 3 consecutive days in STZ rats (oneadministration per day), the glucose, neutral fat and cholesterolcontents in blood were determined. Accordingly, STZ and normal rats wereadministered with the vehicle (physiological saline) at identical dailyrate and duration prior to determination of similar hematic indicesmentioned above. In addition, rats administered subcutaneously (s.c.)with insulin (8 U/kg) daily (one administration per day) for 3consecutive days were used as positive controls. The results are shownin FIG. 1 to 3.

Observations

Administration with vitamin K₅ for 3 consecutive days elicited recoveryof changes found in all hematic indices in rats; namely, the glucose,neutral fat and cholesterol contents.

Example 6 Analysis of Partial Amino Acid Sequence of pp140

pp140 purified in Example 1 was isolated by electrophoresis, followed bytranscription in PVDF membrane, treatmented with trypsin and furtherisolated with liquid chromatography. The thus isolated pp140 fragmentwas then sequenced by using the 470A-model automated gas-phase proteinsequences/120A-model PTH analyzer (ABI or Applied Biosystem Inc. Corp.,U.S.A.) and the extensively employed Edman degradation method prior todetermination of its partial amino acid sequence. The sequence isdepicted in Sequence Table 5 to 7.

Example 7 Partial Amino Acid Sequencing of pp140 by the Polymerase ChainReaction (PCR) Method

By using extensively applied methods, various primers were derived fromthe thus isolated partial amino acid fragments, and their respectivecombinations were conducted before the PCR method was employed. Theresults revealed a specifically amplified fragment with an approximatelength of 400 bp.

Example 8 Isolation and Purification of mRNA

During the log growth phase, mRNA was isolated from 3×10⁷ musclemyoblast L6 cells (ATCC strain No., CRL-1458) according to the method ofOkayama et al (Methods in Enzymology, 154, 3 (1987)).

Briefly, after cells were lysed with 5.5 M GTC solution (5.5 M guanidinethiocyanate, 25 mM sodium citrate and 0.5% sodium lauryl sarcosine, thelysate was layered on cesium trifluoroacetate solution (density:1.51)cells lysate and centrifuged at 120,000×g for 20 hr before all the RNAin the pellet was recovered. The RNA sample was passaged through anoligo-dT-cellulose column twice prior to recovery by purification of 106μg poly(A)+RNA.

Example 9 Tissue Distribution of p140 mRNA

From various tissues, poly(A)+RNA was purified according to proceduressimilar to those of Example 8. The respective tissue-derived poly(A)+RNAsamples (each sample: 2 μg) were subjected to agarose-gelelectrophoresis and subsequently transferred through a filter. The 2-kbopen reading frame was labeled and used as the internal control beforeallowed to undergo normal hybridization. Autoradiography was conductedon the specifically bound probe and evaluated by densitometric analyseswith an imaging analyzer. When the incidence of β-actin mRNA was taken100 in the various tissues, relative contents of p140 in tissues areindicated in Table 4.

TABLE 4 Tissue distribution of p140 mRNA rat human heart 100 100 brain240  60 spleen  70 — lungs 210 100 liver 130 100 muscles  40 130 kidneys130  40 testes, 320 — placenta — 220 pancreas — 330 (—): representsexperiments that were not done

Observation

Examination of all the various tissues studied reveals incidences ofmRNA, whose effects are though to radiate over an extensive range oftissues. High incidence of mRNA is found especially in the humanpancreas.

Example 10 Establishing the cDNA Library

A cDNA library was established according to the modified Gubler andHoffman method (Gene 25, 263, (1983)).

From poly(A)+RNA (5 μg) derived in Example 2, a first strand wasconstructed with the reverse transcription enzyme, followed bytransformation of a second strand with EcoRI adaptor ligation beforeexcess adaptors and primers were eliminated by gel filtration columnchromatography (Sephacryl S-500HR column; Pharmacia Corp.). Theremaining 1,620 ng of CDNA fraction was subsequently recovered.

The above construction procedures for cDNA library were accomplishedwith a λgt 10 cloning system kit (Amersham Corp.).

Next, the λgt 10 phage (Amersham Corp.) and λZAPII phage (Stra TageneCorp.) were ligated at the EcoRI-treated arms of 1.8-kb mean length. Aphage cDNA library of an independent count of 3×10₅ was established.

Example 11 Cloning and Sequencing

Based on the phage DNA library established in Example 10, clones wereduplicated to approximately 1×10₅ plagues/plate. The approximately400-bp fragments harvested in Example 7 were designated as probes beforescreening was conducted. Of the positive controls, subcloning of longstrands of the inserts in EcoRI side of plasmid vector pGEM-3Zf(+) (3199bp; Promega Corp.) was established. T7 or SP6 was sequenced as theprimer.

DNA sequencing based on the dideoxy terminator method was performedaccording to the cyclo-sequencing method using fluorescent di-terminator(ABI, USA). Furthermore, sequence reading was realized with a DNAsequencer (Model 373A; ABI, USA).

As such, nucleotide sequences of mean 300 bases were established from 5′or 3′ side of the respective clone.

Example 12 Partial Sequence Analysis

When the nucleotide sequence from Example 11 was subjected to a homologysearch with all the nucleotide sequences stored in previously registereddata base (GenBank and EMBL) with the FASTA program of Lipman andPearson, the sequenced clones would identify clones containing novelsequences. Nucleotide sequences of the identified clone were convertedto amino acid sequences based on 3 possibly constructed frames.

Additionally, novel amino acid sequences in the amino acid sequenceswere also revealed.

However, the DNA clone that has cloned does not necessarily cover thewhole mRNA length. In such a case, the clone is most unlikely to containthe N terminal of amino acid sequence.

As such, the Northern analysis was used to determine if the whole lengthof the established clone was complemented. In other words, thepoly(A)+RNA, isolated from Example 8→Example 9 procedures byelectrophoresis, was blotted on a nylon membrane. When the subclonedcDNA insert was hybridized as a probe, a single band at approximately4400-bp position was observed. Since sizes of the clones wereapproximated to 2200 bp, PCR was performed at the 5′ and 3′ sides toread the whole cDNA length with the 3′-RACE (BRL Corp.) system and5′-RACE (CLONTECH Corp.) system kits.

Example 13 Determining the Sequence and Open Reading Frame of Whole cDNALength

Random sequencing of the whole length of cDNA sequence was appropriatedaccording to the method of Sambrook et al. (Molecular Cloning: ed.Sambrook J, Fritsch EF, Maniatis T; 1989, Cold Spring Harbor LaboratoryPress).

Briefly, plasmid was recovered from the clone and the isolated cDNAinsert was then purified before ligation and fragmentation. The terminalof DNA fragment was further smoothened by T4 polymerase and DNAfragments of approximated 400-bp length were recovered by agaroseelectrophoresis. DNA fragments thus established were subjected tocloning in the Smal side of plasmid vector and pGEM-3Zf(+) (3199 bp;Promega Corp.) before transformation in E. Coli. Eighty colonies werepicked up at random and plasmid DNAs were refined prior to DNAsequencing of these 20 plasmids (possessing cDNA fragments as inserts).DNA sequencing and sequence reading were performed according to themethod described in Example 11. Sequence data of cDNA fragments wereconstructed to the linkage sequences with the DNA sequence program ofDNASIS. The basic sequence portaryed in Seq. ID No. 3 was henceconstructed. From sequence data of the whole cDNA length, the openreading frame (ORF) was determined. The amino acid sequence was furthertranslated and the sequence thus established is illustrated in Seq.No. 1. One of the frames possesses the 2993-bp ORF, that wasapproximated to 3,000 bp of the whole ORF length of the Eck family.Therefore, the said polypeptide in the present invention is postulatedto possess a whole length of 2,993 bp.

Based on its hydrophobicity, protein p140 was further postulated to be atypical Type I membrane protein (FIG. 4 demarcates the zone with eitherhigh (+) or low (−) hydrophobicity).

All in all, the said p140 polypeptide is a typical membrane protein with993 amino acids and the length of its ORF is 2982 bp. Furthermore, theestimated molecular weight of the said p140 polypeptide is 109,860 Da,and is 140 kD when evaluated from the bonds of its polysaccharide chain.

Example 14 Construction of Plasmid Vector for Using the Preparation ofExpression Vector

As an expression vector, pUC-SRαML-1 (This vector is disclosed itselfand preparation thereof in European Patent publication No. 559428)derivative was used. This derivative was constructed to insert twokinds of fragments as shown below:

fragment T7 5′ GTAATACGACTCACTATAGGGGAGAGCT 3′ (SEQ ID No. 8)

3′ ACGTCATTATGCTGAGTGATATCCCCTC 5′ (SEQ ID No. 9)

between PstI and SacI and

fragment SP6 5′ CTAGTCTATAGTGTCACCTAAATCGTGGGTAC 3′ (SEQ ID No. 10)

3′ AGATATCACAGTGGATTTAGCAC 5′ (SEQ ID No. 11)

between SpeI and KpnI site in the multi-cloning site, respectively.

The pUC-SRαML1 vector was digested with PstI and SacI and the resultingdigest was subjected to agarose gel electrophoresis to prepare andrecover an about 4.1 kbp fragment and thereafter removing the 5′-endphosphoric acid group by BAP (bacterial alkaline phosphatase) treatment.The phosphorylated DNA fragment T7 was ligated with the thus preparedabout 4.1 kbp fragment from pUC-SRαML1 to make them into a circularform. The resulting vector was, moreover, digested with SpeI and KpnIand the resulting digest was subjected to agarose gel electrophoresis toprepare and recover an about 4.1 kbp fragment and thereafter removingthe 5′-end phosphoric acid group by BAP (bacterial alkaline phosphatase)treatment. The phosphorylated DNA fragment SP6 was ligated with the thusprepared about 4.1 kbp fragment to make them into a circular form. Theplasmid vector constructed in this manner was named pUC-SRαML2 (See FIG.3).

Example 15 Construction of Expression Vector

The primers X, Y and YH, that aneal to rat p140 cDNA, were synthesized.Sequences of primers X, Y and YH are as follows:

Primer X

5′- A ATA TAG TCG ACC ACC ATG GAG AAC CCC TAC GTT GGG CGA GCG A -3′ (SEQID No. 12)

Primer Y

5′- CGG CGG ACT AGT TCA GAC CTG CAC GGG CAG TGT CTG G -3′ (SEQ ID No.13)

Primer YH

5′- GCC GCC ACT AGT TCA GTG GTG GTG GTG GTG GTG GAC CTG CAC GGG CAG TGTCTG G -3′ (SEQ ID No. 14)

The plasmid containing cDNA of p140 was subjected to PCR using the thussynthesized oligonucleotides X and Y as templates. The thus obtained PCRfragment contains a sequence placed 5′-adjacent to the initiation codon,that is corresponding to Cozac sequence which is known among skilled inthe art, and cDNA which encodes a protein molecule consisting of thep140 protein. The PCR fragment was digested with SaII-SpeI and theresulting digest was separated and purified and then inserted into theSaII-SpeI site of the pUC-SRαML2 prepared in Example 14 to obtain anexpression vector pUC-SRαML2-p140-A.

Moreover, the plasmid containing cDNA of p140 was subjected to PCR usingthe synthesized oligonucleotides X and YH as templates. The thusobtained PCR fragment contains a sequence placed 5′-adjacent to theinitiation codon, that is corresponding to Cozac sequence which is knownamong skilled in the art, and cDNA which encodes a protein moleculeconsisting of the p140 protein and six additional histidine (His)residues attached to its C-terminal end. The PCR fragment was digestedwith SaII-SpeI and the resulting digest was separated and purified andthen inserted into the SaII-SpeI site of the pUC-SRαML2 prepared inExample 14 to obtain an expression vector pUC-SRαML2-p140-B.

Moreover, primer Z and ZH were synthesized. Sequences of primer Z and ZHare as follows: (These were adjoined to amino-terminal end oftransmembrane region in cDNA.)

Primer Z

5′- CGG CGG ACT AGT TCA TGA GCC TCT TTC ACT CGT GGT CTC AAA CT -3′ (SEQID No. 15)

Primer ZH

5′- GCC GCC ACT AGT TCA GTG GTG GTG GTG GTG GTG TGA GCC TCT TTC ACT CGTGGT CTC AAA CT -3′ (SEQ ID No. 16)

The plasmid containing cDNA of p140 was subjected to PCR using the thussynthesized oligonucleotides X and Z as templates. The thus obtained PCRfragment contains a sequence placed 5′-adjacent to the initiation codon,that is corresponding to Cozac sequence which is known among skilled inthe art, and cDNA which encodes a polypeptide consisting of the p140protein extracellular part. The PCR fragment was digested with SaII andNotI and the resulting digest was separated and purified and theninserted into the SaII-SpeI site of the pUC-SRαML2 prepared in Example14 to obtain an expression vector pUC-SRαML2-p140-C.

Moreover, the plasmid containing cDNA of p140 was subjected to PCR usingthe synthesized oligonucleotides X and ZH as templates. The thusobtained PCR fragment contains a sequence placed 5′-adjacent to theinitiation codon, that is corresponding to Cozac sequence which is knownamong skilled in the art, and cDNA which encodes a polypeptideconsisting of the p140 protein extracellular part and six additionalhistidine (His) residues attached to its C-terminal end. The PCRfragment was digested with SaII-SpeI and the resulting digest wasseparated and purified and then inserted into the SaII-SpeI site of thepUC-SRαML2 prepared in Example 14 to obtain an expression vectorpUC-SRαML2-p140-D.

Each of the thus constructed pUC-SRαML2-p140-A, pUC-SRαML2-p140-B,pUC-SRαML2-p140-C and pUC-SRαML2-p140-D plasmids were transfected intoan E. coli strain DH5, recovered from a 100 ml culture of the resultingtransformant and then purified by CsCI density gradient centrifugationtwice.

Example 16 Expression in COS Cells

Each of the plasmid DNA preparations pUC-SRαML2, pUC-SRαML2-p140-A,pUC-SRαML2-p140-B, pUC-SRαML2-p140-C and pUC-SRαML2-p140-D wereintroduced into COS-7 cells (Cell, 23, 175 (1981)) by means of thediethylaminoethyl (DEAE) dextran method (J. Immunology, 136, 4291(1986)).

That is, about 1.8×10₆ COS-7 cells were inoculated into a 225 cm²capacity flask (manufactured by Corning) together with 50 ml of a liquidculture medium (Dulbecco's modified MEM medium supplemented with 10%decomplemented fetal bovine serum). After overnight incubation in acarbon dioxide incubator (37° C., 5% CO₂) and subsequent removal of theculture supernatant, 12 ml of a DNA cocktail (Dulbecco's modified MEMmedium supplemented with 15 4g of each plasmid DNA, 50 mM Tris-HCIbuffer (pH 7.4) and 400 μg/ml of DEAE-dextran) was added to each flaskand culture was carried out for 3 hours at 37° C. in an atmosphere of 5%CO₂. Thereafter, the DNA cocktail was replaced by 15 ml of a chloroquinesolution (Dulbecco's modified MEM medium supplemented with 150 μMchloroquine and 7% decomplemented fetal bovine serum), followed byadditional 3 hours of culture.

After removing the chloroquine solution, the aforementioned liquidculture medium (50 ml) was added to each of the resulting flasks whichwere then incubated at 37° C. in an atmosphere of 5% CO₂ for 72 hours tofind growth of the cells in each flask into almost monolayer form. Afterremoving the culture supernatant, the cells in each flask were washedwith a serum-free liquid culture medium (trade name, SFM-101; availablefrom Nissui Pharmaceutical Co., Ltd.) and then supplied with 75 ml ofthe same serum-free liquid culture medium, and the culturing wascontinued for another 72 hours. Thereafter, the resulting culturesupernatants were recovered and cells were lysed as represented inExample 1. These supernatants and cell lysates were filtered through amembrane filter (trade name, STERIVEX-GS; available from MilliporeCorp.) to remove cell debris. The thus obtained culture supernatantsamples were stored at 4° C. for future use. A The cell lysates of COScells which have been transformed with plasmid containing thepUC-SRαML2-p140-A and pUC-SRαML2-p140-B inserts are expected to containexpressed mature protein moieties of polypeptides which correspond top140 protein. And culture supernatants of COS cells which have beentransformed with plasmid containing the pUC-SRαML2-p140-C andpUC-SRαML2-p140-D inserts are expected to contain secreted polypeptideswhich correspond to p140 protein extracellular part.

Example 17 Confirmation of Expression

A 2 ml portion of each of the culture supernatants of transformed COScells obtained in Example 16 was concentrated to a volume of 100 mlusing a centrifugal concentration filter (trade name, Centricon-10;available from Millipore Corp.). A 1 μl portion of each of the thusconcentrated samples was mixed with the same volume of a loading buffer(0.125 M Tris-HCI buffer (pH 6.8), 4% sodium dodecyl sulfate and 30%glycerol) for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gelelectrophoresis) use, and the mixture was treated at 90° C. for 3minutes and then subjected to SDS-PAGE.

In the case of the pUC-SRαML2-p140-B and pUC-SRαML2-p140-D proteinshaving His hexamer introduced to the C-terminus of the proteins, notonly their corresponding cell lysates and COS cell culture supernatantsbut also their purified products were subjected to the SDS-PAGEanalysis.

Purification of the protein was carried out by means of a metal chelateaffinity chromatography (Biotechnology, 9, 273, (1991)), making use ofthe function of His to form complex compounds with various transitionmetal ions. That is, a culture supernatant (350 ml) or cell lysates (100ml) obtained from COS cells was mixed with a sodium chloride aqueoussolution in such an amount that the final concentration of the saltbecame 1 M, and the resulting mixture was applied to a column packedwith 4 ml of a zinc-linked chelating Sepharose (trade name, ChelatingSepharose Fast-Flow; available from Pharmacia) to adsorb the protein tothe resin. The column was washed with 50 mM phosphate buffer (pH 7.0)containing 1 M sodium chloride aqueous solution (40 ml), and the proteinretained in the column was eluted with 50 mM phosphate buffer (pH 7.0)containing 1 M sodium chloride aqueous solution and 0.4 M imidazole.Thereafter, the resulting elute was concentrated to a volume of 100 1μl, and a portion of the concentrated sample was subjected to SDS-PAGEanalysis.

The SDS-PAGE analysis was carried out using a SDS 10/20 gradient gel anda product which corresponds to a molecular weight of p140 was detectedin samples prepared from COS cells transfected pUC-SRαML2-p140-A andp140-B. Furthermore, a polypeptide which corresponds to a molecularweight of extracellular portion of p140 was detected in untreated andpurified supernatants, not cell lysates, prepared from COS cellstransfected pUC-SRαML2-p140-C and p140-D.

Formulation Example 1

The following components were admixed in convention method and punchedout to obtain 100 tablets each containing 5 mg of active ingredient.

Vitamin K₅ 500.0 mg Carboxymethylcellulose calcium 200.0 mg Magnesiumstearate 100.0 mg Microcrystalline cellulose 9.2 mg

16 993 amino acids amino acid linear protein rat skeletal musclemyoblast L6 1 Met Glu Asn Pro Tyr Val Gly Arg Ala Arg Ala Ala Ala GluArg Ala 1 5 10 15 Ala Ala Glu Ala Thr Asn Ser Leu Ser Ile Leu Val ArgPro Thr Ser 20 25 30 Glu Gly Ser Arg Ile Asp Ser Glu Phe Val Glu Leu AlaTrp Thr Ser 35 40 45 His Pro Glu Ser Gly Trp Glu Glu Val Ser Ala Tyr AspGlu Ala Met 50 55 60 Asn Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Arg GluSer Ser Gln 65 70 75 80 Asn Asn Trp Leu Arg Thr Gly Phe Ile Trp Arg ArgGlu Val Gln Arg 85 90 95 Val Tyr Val Glu Leu Lys Phe Thr Val Arg Asp CysAsn Ser Ile Pro 100 105 110 Asn Ile Pro Gly Ser Cys Lys Glu Thr Phe AsnLeu Phe Tyr Tyr Glu 115 120 125 Ala Asp Ser Asp Val Ala Ser Ala Ser SerPro Phe Trp Met Glu Asn 130 135 140 Pro Tyr Val Lys Val Asp Thr Ile AlaPro Asp Glu Ser Phe Ser Arg 145 150 155 160 Leu Asp Ala Gly Arg Val AsnThr Lys Val Arg Ser Phe Gly Pro Leu 165 170 175 Ser Lys Ala Gly Phe TyrLeu Ala Phe Gln Asp Gln Gly Ala Cys Met 180 185 190 Ser Leu Ile Ser ValArg Ala Phe Tyr Lys Lys Cys Ala Ser Thr Thr 195 200 205 Ala Gly Phe AlaLeu Phe Pro Glu Thr Leu Thr Gly Ala Glu Pro Thr 210 215 220 Ser Leu ValIle Ala Pro Gly Thr Cys Ile Ala Asn Ala Val Glu Val 225 230 235 240 SerVal Pro Leu Lys Leu Tyr Cys Asn Gly Asp Gly Glu Trp Met Val 245 250 255Pro Val Gly Ala Cys Thr Cys Ala Thr Gly His Glu Pro Ala Ala Lys 260 265270 Glu Thr Gln Cys Arg Ala Cys Pro Pro Gly Ser Tyr Lys Ala Lys Gln 275280 285 Gly Glu Gly Pro Cys Leu Pro Cys Pro Pro Asn Ser Arg Thr Thr Ser290 295 300 Pro Ala Ala Ser Ile Cys Thr Cys His Asn Asn Phe Tyr Arg AlaAsp 305 310 315 320 Ser Asp Thr Ala Asp Ser Ala Cys Thr Thr Val Pro SerPro Pro Arg 325 330 335 Gly Val Ile Ser Asn Val Asn Glu Thr Ser Leu IleLeu Glu Trp Ser 340 345 350 Glu Pro Arg Asp Leu Gly Gly Arg Asp Asp LeuLeu Tyr Asn Val Ile 355 360 365 Cys Lys Lys Cys Arg Gly Ser Ser Gly AlaGly Gly Pro Ala Thr Cys 370 375 380 Ser Arg Cys Asp Asp Asn Val Glu PheGlu Pro Arg Gln Leu Gly Leu 385 390 395 400 Thr Glu Arg Arg Val His IleSer His Leu Leu Ala His Thr Arg Tyr 405 410 415 Thr Phe Glu Val Gln AlaVal Asn Gly Val Ser Gly Lys Ser Pro Leu 420 425 430 Pro Pro Arg Tyr AlaAla Val Asn Ile Thr Thr Asn Gln Ala Ala Pro 435 440 445 Ser Glu Val ProThr Leu His Leu His Ser Ser Ser Gly Ser Ser Leu 450 455 460 Thr Leu SerTrp Ala Pro Pro Glu Arg Pro Asn Gly Val Ile Leu Asp 465 470 475 480 TyrGlu Met Lys Tyr Phe Glu Lys Ser Lys Gly Ile Ala Ser Thr Val 485 490 495Thr Ser Gln Lys Asn Ser Val Gln Leu Asp Gly Leu Gln Pro Asp Ala 500 505510 Arg Tyr Val Val Gln Val Arg Ala Arg Thr Val Ala Gly Tyr Gly Gln 515520 525 Tyr Ser Arg Pro Ala Glu Phe Glu Thr Thr Ser Glu Arg Gly Ser Gly530 535 540 Ala Gln Gln Leu Gln Glu Gln Leu Pro Leu Ile Val Gly Ser ThrVal 545 550 555 560 Ala Gly Phe Val Phe Met Val Val Val Val Val Ile AlaLeu Val Cys 565 570 575 Leu Arg Lys Gln Arg Gln Gly Pro Asp Ala Glu TyrThr Glu Lys Leu 580 585 590 Gln Gln Tyr Val Ala Pro Arg Met Lys Val TyrIle Asp Pro Phe Thr 595 600 605 Tyr Glu Asp Pro Asn Glu Ala Val Arg GluPhe Ala Lys Glu Ile Asp 610 615 620 Val Ser Cys Val Lys Ile Glu Glu ValIle Gly Ala Gly Glu Phe Gly 625 630 635 640 Glu Val Cys Arg Gly Arg LeuLys Leu Pro Gly Arg Arg Glu Val Phe 645 650 655 Val Ala Ile Lys Thr LeuLys Val Gly Tyr Thr Glu Arg Gln Arg Arg 660 665 670 Asp Phe Leu Ser GluAla Ser Ile Met Gly Gln Phe Asp His Pro Asn 675 680 685 Ile Ile Arg LeuGlu Gly Val Val Thr Lys Ser Arg Pro Val Met Ile 690 695 700 Leu Thr GluPhe Met Glu Asn Cys Ala Leu Asp Ser Phe Leu Arg Leu 705 710 715 720 AsnAsp Gly Gln Phe Thr Val Ile Gln Leu Val Gly Met Leu Arg Gly 725 730 735Ile Ala Ala Gly Met Lys Tyr Leu Ser Glu Met Asn Tyr Val His Arg 740 745750 Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys 755760 765 Val Ser Asp Phe Gly Leu Ser Arg Phe Leu Glu Asp Asp Pro Ser Asp770 775 780 Pro Thr Tyr Thr Ser Ser Leu Gly Gly Lys Ile Pro Ile Arg TrpThr 785 790 795 800 Ala Pro Glu Ala Ile Asp Tyr Arg Lys Phe Thr Ser AlaSer Asp Val 805 810 815 Trp Ser Tyr Gly Ile Val Met Trp Glu Val Met SerTyr Gly Glu Arg 820 825 830 Pro Tyr Trp Asp Met Ser Asn Gln Asp Val IleAsn Ala Val Glu Gln 835 840 845 Asp Tyr Arg Leu Pro Pro Pro Met Asp CysPro Ala Ala Leu His Gln 850 855 860 Leu Met Leu Asp Cys Trp Val Arg AspArg Asn Leu Arg Pro Lys Phe 865 870 875 880 Ser Gln Ile Val Asn Thr LeuAsp Lys Leu Ile Arg Asn Ala Ala Ser 885 890 895 Leu Lys Val Ile Ala SerAla Pro Ser Gly Met Ser Gln Pro Leu Leu 900 905 910 Asp Arg Thr Val ProAsp Tyr Thr Thr Phe Thr Thr Val Gly Asp Trp 915 920 925 Leu Asp Ala IleLys Met Gly Arg Tyr Lys Glu Ser Phe Val Gly Ala 930 935 940 Gly Phe AlaSer Phe Asp Leu Val Ala Gln Met Thr Ala Glu Asp Leu 945 950 955 960 LeuArg Ile Gly Val Thr Leu Ala Gly His Gln Lys Lys Ile Leu Ser 965 970 975Ser Ile Gln Asp Met Arg Leu Gln Met Asn Gln Thr Leu Pro Val Gln 980 985990 Val 2982 base pairs nucleic acid single linear cDNA to mRNA ratskeletal muscle myoblast L6 2 ATGGAGAACC CCTACGTTGG GCGAGCGAGAGCAGCAGCGG AGCGAGCAGC GGCAGAAGCC 60 ACGAATTCAC TATCGATCCT GGTTCGGCCCACCTCTGAAG GTTCCAGAAT CGATAGTGAA 120 TTCGTGGAGC TGGCATGGAC ATCTCATCCAGAGAGTGGGT GGGAAGAAGT GAGCGCCTAC 180 GATGAAGCCA TGAATCCTAT CCGCACGTATCAGGTGTGTA ACGTGCGCGA GTCCAGCCAG 240 AACAACTGGC TGCGGACCGG TTTCATCTGGCGGCGGGAAG TCCAGCGCGT CTACGTGGAG 300 CTGAAGTTTA CCGTGAGAGA TTGCAACAGCATCCCCAACA TCCCTGGCTC CTGCAAGGAA 360 ACCTTCAACC TTTTTTACTA CGAGGCTGATAGCGATGTGG CGTCAGCCTC CTCTCCCTTC 420 TGGATGGAGA ACCCCTACGT GAAAGTGGACACCATTGCGC CAGATGAGAG CTTCTCGCGG 480 CTAGACGCTG GGCGCGTTAA CACCAAAGTGCGCAGCTTCG GGCCGCTTTC CAAAGCCGGC 540 TTCTACTTGG CCTTCCAGGA CCAGGGTGCCTGCATGTCAC TCATCTCTGT GCGCGCCTTC 600 TACAAGAAGT GTGCATCCAC CACTGCAGGCTTCGCACTCT TCCCCGAGAC CCTCACGGGG 660 GCTGAGCCCA CTTCGCTGGT CATTGCCCCTGGCACCTGCA TCGCTAACGC TGTGGAGGTG 720 TCTGTACCGC TCAAGCTCTA CTGCAATGGCGACGGGGAGT GGATGGTGCC CGTTGGTGCC 780 TGCACCTGCG CTACTGGCCA TGAGCCAGCCGCCAAGGAGA CCCAGTGCCG CGCCTGTCCC 840 CCTGGGAGCT ACAAGGCAAA GCAAGGAGAGGGGCCCTGCC TCCCCTGTCC CCCCAATAGC 900 CGCACCACCT CGCCGGCTGC CAGCATCTGCACCTGTCACA ATAATTTCTA CCGCGCAGAC 960 TCAGACACAG CGGACAGCGC CTGCACCACGGTGCCGTCTC CCCCCCGGGG TGTGATCTCC 1020 AATGTGAATG AGACCTCGCT GATCCTCGAGTGGAGTGAGC CCCGGGACCT TGGCGGACGA 1080 GATGACCTCC TTTATAATGT TATCTGTAAGAAGTGCCGTG GCAGCTCTGG GGCTGGAGGT 1140 CCGGCGACCT GTTCACGCTG TGATGACAACGTGGAGTTCG AGCCCCGACA GCTGGGCCTG 1200 ACCGAGCGCC GGGTCCACAT CAGCCACCTGTTGGCCCACA CCCGCTACAC CTTTGAGGTG 1260 CAGGCTGTCA ACGGCGTCTC TGGCAAAAGCCCTTTGCCGC CCCGCTATGC AGCTGTGAAT 1320 ATCACCACCA ACCAGGCCGC CCCATCAGAAGTGCCTACGC TCCACTTGCA CAGCAGTTCA 1380 GGGAGCAGCC TGACCCTGTC CTGGGCACCCCCGGAGCGGC CTAACGGAGT CATCTTGGAC 1440 TATGAGATGA AGTACTTTGA GAAGAGTAAAGGCATCGCCT CCACTGTCAC CAGCCAGAAG 1500 AACTCTGTAC AACTGGACGG ACTGCAGCCCGACGCCCGCT ATGTAGTTCA GGTCCGGGCT 1560 CGCACAGTAG CAGGTTACGG ACAGTATAGCCGCCCAGCTG AGTTTGAGAC CACGAGTGAA 1620 AGAGGCTCAG GGGCCCAGCA GCTTCAAGAGCAGCTTCCCC TAATTGTGGG ATCCACCGTA 1680 GCTGGCTTTG TCTTCATGGT GGTCGTCGTGGTCATTGCTC TTGTCTGCCT CAGGAAGCAG 1740 CGCCAGGGCC CTGATGCAGA ATACACGGAGAAGTTGCAGC AATACGTTGC CCCCAGGATG 1800 AAAGTTTACA TTGACCCCTT TACCTACGAGGATCCCAATG AGGCCGTCCG AGAGTTCGCC 1860 AAGGAGATCG ATGTGTCCTG CGTCAAGATCGAGGAGGTGA TTGGAGCTGG GGAGTTTGGG 1920 GAAGTGTGCC GGGGTCGGCT GAAACTGCCCGGCCGCCGGG AGGTGTTCGT GGCCATCAAG 1980 ACACTGAAGG TGGGATACAC GGAGAGGCAGCGGCGGGACT TCCTGAGTGA GGCTTCCATC 2040 ATGGGTCAAT TTGACCATCC AAATATAATCCGTCTAGAGG GCGTGGTCAC CAAAAGTCGT 2100 CCAGTCATGA TCCTCACTGA GTTCATGGAGAACTGTGCCC TGGACTCCTT CCTACGGCTC 2160 AATGACGGGC AGTTCACAGT CATCCAGCTTGTGGGCATGT TGCGTGGCAT TGCTGCCGGC 2220 ATGAAGTACT TGTCTGAGAT GAACTACGTGCACCGTGACC TCGCTGCCCG CAACATCCTT 2280 GTCAACAGTA ACTTGGTCTG CAAAGTATCTGACTTTGGGC TCTCCCGCTT CCTGGAGGAC 2340 GACCCCTCAG ACCCCACCTA CACCAGCTCCCTGGGTGGGA AGATCCCTAT CCGTTGGACC 2400 GCCCCAGAGG CCATAGACTA TCGGAAGTTCACGTCTGCCA GCGATGTCTG GAGCTACGGG 2460 ATCGTCATGT GGGAGGTCAT GAGCTACGGAGAGCGACCAT ACTGGGACAT GAGCAACCAG 2520 GATGTCATCA ATGCCGTAGA GCAAGACTATCGGTTACCAC CCCCCATGGA CTGCCCAGCG 2580 GCGCTGCACC AGCTCATGCT GGACTGTTGGGTGCGGGACC GGAACCTCAG GCCCAAGTTC 2640 TCCCAAATCG TCAACACGCT AGACAAGCTTATCCGCAATG CTGCCAGCCT CAAGGTCATC 2700 GCCAGTGCCC CATCTGGCAT GTCCCAGCCCCTCCTAGACC GCACGGTCCC AGATTATACG 2760 ACCTTCACGA CGGTGGGCGA CTGGCTAGATGCCATCAAGA TGGGGAGGTA TAAAGAGAGC 2820 TTCGTCGGTG CGGGTTTTGC CTCCTTTGACCTGGTGGCCC AGATGACTGC AGAAGATCTG 2880 CTAAGGATCG GGGTCACTTT GGCCGGCCACCAGAAGAAGA TCCTCAGCAG TATCCAGGAC 2940 ATGCGGCTGC AGATGAACCA GACACTGCCCGTGCAGGTCT GA 2982 4027 base pairs nucleic acid single linear cDNA tomRNA rat skeletal muscle myoblast L6 3 GAAAAATGAA GATCTATACC GACAGCAGATCAGTGGCTGC CTGGGGCAAA GTTGGAGGGA 60 CATGTTATTT TGATTGTGAT GACATAATACATGCAAACAC GGCTAATCCT CTCAAAGCAT 120 ACACTTATAC ATGTGCAGCT TGGTATACATAAATTATCCA TTACAAAACT ATGAGAAAGC 180 TATCACCACT ATGAAGCACC ACTCACAGTATGTGAATCTC CACCCCCCTT CCACTGCTGA 240 GACACAGAAA TCCTAGACTG GATGGAGAACCCCTACGTTG GGCGAGCGAG AGCAGCAGCG 300 GAGCGAGCAG CGGCAGAAGC CACGAATTCACTATCGATCC TGGTTCGGCC CACCTCTGAA 360 GGTTCCAGAA TCGATAGTGA ATTCGTGGAGCTGGCATGGA CATCTCATCC AGAGAGTGGG 420 TGGGAAGAAG TGAGCGCCTA CGATGAAGCCATGAATCCTA TCCGCACGTA TCAGGTGTGT 480 AACGTGCGCG AGTCCAGCCA GAACAACTGGCTGCGGACCG GTTTCATCTG GCGGCGGGAA 540 GTCCAGCGCG TCTACGTGGA GCTGAAGTTTACCGTGAGAG ATTGCAACAG CATCCCCAAC 600 ATCCCTGGCT CCTGCAAGGA AACCTTCAACCTTTTTTACT ACGAGGCTGA TAGCGATGTG 660 GCGTCAGCCT CCTCTCCCTT CTGGATGGAGAACCCCTACG TGAAAGTGGA CACCATTGCG 720 CCAGATGAGA GCTTCTCGCG GCTAGACGCTGGGCGCGTTA ACACCAAAGT GCGCAGCTTC 780 GGGCCGCTTT CCAAAGCCGG CTTCTACTTGGCCTTCCAGG ACCAGGGTGC CTGCATGTCA 840 CTCATCTCTG TGCGCGCCTT CTACAAGAAGTGTGCATCCA CCACTGCAGG CTTCGCACTC 900 TTCCCCGAGA CCCTCACGGG GGCTGAGCCCACTTCGCTGG TCATTGCCCC TGGCACCTGC 960 ATCGCTAACG CTGTGGAGGT GTCTGTACCGCTCAAGCTCT ACTGCAATGG CGACGGGGAG 1020 TGGATGGTGC CCGTTGGTGC CTGCACCTGCGCTACTGGCC ATGAGCCAGC CGCCAAGGAG 1080 ACCCAGTGCC GCGCCTGTCC CCCTGGGAGCTACAAGGCAA AGCAAGGAGA GGGGCCCTGC 1140 CTCCCCTGTC CCCCCAATAG CCGCACCACCTCGCCGGCTG CCAGCATCTG CACCTGTCAC 1200 AATAATTTCT ACCGCGCAGA CTCAGACACAGCGGACAGCG CCTGCACCAC GGTGCCGTCT 1260 CCCCCCCGGG GTGTGATCTC CAATGTGAATGAGACCTCGC TGATCCTCGA GTGGAGTGAG 1320 CCCCGGGACC TTGGCGGACG AGATGACCTCCTTTATAATG TTATCTGTAA GAAGTGCCGT 1380 GGCAGCTCTG GGGCTGGAGG TCCGGCGACCTGTTCACGCT GTGATGACAA CGTGGAGTTC 1440 GAGCCCCGAC AGCTGGGCCT GACCGAGCGCCGGGTCCACA TCAGCCACCT GTTGGCCCAC 1500 ACCCGCTACA CCTTTGAGGT GCAGGCTGTCAACGGCGTCT CTGGCAAAAG CCCTTTGCCG 1560 CCCCGCTATG CAGCTGTGAA TATCACCACCAACCAGGCCG CCCCATCAGA AGTGCCTACG 1620 CTCCACTTGC ACAGCAGTTC AGGGAGCAGCCTGACCCTGT CCTGGGCACC CCCGGAGCGG 1680 CCTAACGGAG TCATCTTGGA CTATGAGATGAAGTACTTTG AGAAGAGTAA AGGCATCGCC 1740 TCCACTGTCA CCAGCCAGAA GAACTCTGTACAACTGGACG GACTGCAGCC CGACGCCCGC 1800 TATGTAGTTC AGGTCCGGGC TCGCACAGTAGCAGGTTACG GACAGTATAG CCGCCCAGCT 1860 GAGTTTGAGA CCACGAGTGA AAGAGGCTCAGGGGCCCAGC AGCTTCAAGA GCAGCTTCCC 1920 CTAATTGTGG GATCCACCGT AGCTGGCTTTGTCTTCATGG TGGTCGTCGT GGTCATTGCT 1980 CTTGTCTGCC TCAGGAAGCA GCGCCAGGGCCCTGATGCAG AATACACGGA GAAGTTGCAG 2040 CAATACGTTG CCCCCAGGAT GAAAGTTTACATTGACCCCT TTACCTACGA GGATCCCAAT 2100 GAGGCCGTCC GAGAGTTCGC CAAGGAGATCGATGTGTCCT GCGTCAAGAT CGAGGAGGTG 2160 ATTGGAGCTG GGGAGTTTGG GGAAGTGTGCCGGGGTCGGC TGAAACTGCC CGGCCGCCGG 2220 GAGGTGTTCG TGGCCATCAA GACACTGAAGGTGGGATACA CGGAGAGGCA GCGGCGGGAC 2280 TTCCTGAGTG AGGCTTCCAT CATGGGTCAATTTGACCATC CAAATATAAT CCGTCTAGAG 2340 GGCGTGGTCA CCAAAAGTCG TCCAGTCATGATCCTCACTG AGTTCATGGA GAACTGTGCC 2400 CTGGACTCCT TCCTACGGCT CAATGACGGGCAGTTCACAG TCATCCAGCT TGTGGGCATG 2460 TTGCGTGGCA TTGCTGCCGG CATGAAGTACTTGTCTGAGA TGAACTACGT GCACCGTGAC 2520 CTCGCTGCCC GCAACATCCT TGTCAACAGTAACTTGGTCT GCAAAGTATC TGACTTTGGG 2580 CTCTCCCGCT TCCTGGAGGA CGACCCCTCAGACCCCACCT ACACCAGCTC CCTGGGTGGG 2640 AAGATCCCTA TCCGTTGGAC CGCCCCAGAGGCCATAGACT ATCGGAAGTT CACGTCTGCC 2700 AGCGATGTCT GGAGCTACGG GATCGTCATGTGGGAGGTCA TGAGCTACGG AGAGCGACCA 2760 TACTGGGACA TGAGCAACCA GGATGTCATCAATGCCGTAG AGCAAGACTA TCGGTTACCA 2820 CCCCCCATGG ACTGCCCAGC GGCGCTGCACCAGCTCATGC TGGACTGTTG GGTGCGGGAC 2880 CGGAACCTCA GGCCCAAGTT CTCCCAAATCGTCAACACGC TAGACAAGCT TATCCGCAAT 2940 GCTGCCAGCC TCAAGGTCAT CGCCAGTGCCCCATCTGGCA TGTCCCAGCC CCTCCTAGAC 3000 CGCACGGTCC CAGATTATAC GACCTTCACGACGGTGGGCG ACTGGCTAGA TGCCATCAAG 3060 ATGGGGAGGT ATAAAGAGAG CTTCGTCGGTGCGGGTTTTG CCTCCTTTGA CCTGGTGGCC 3120 CAGATGACTG CAGAAGATCT GCTAAGGATCGGGGTCACTT TGGCCGGCCA CCAGAAGAAG 3180 ATCCTCAGCA GTATCCAGGA CATGCGGCTGCAGATGAACC AGACACTGCC CGTGCAGGTC 3240 TGACGCTCAG CTCCAGCGAG GGGCGTGGCCCCCCGGGACT GCACAAGGAT TCTGACCAGC 3300 CAGCTGGACT TTTGGATACC TGGCCTTTGGCTGTGGCCCA GAAGACAGAA GTTCGGGGGA 3360 GAACCCTAGC TGTGACTTCT CCAAGCCTGTGCTCCCTCCC AGGAAGTGTG CCCCAAACCT 3420 CTTCATATTG AAGATGGATT AGAAGAGGGGGTGATATCCC CTCCCCAGAT GCCTCAGGGC 3480 CCAGGCCTGC CTGCTCTCCA GTCGGGGATCTTCACAACTC AGATTTGGTT GTGCTTCAGT 3540 AGTGGAGGTC CTGGTAGGGT CGGGTGGGGATAAGCCTGGG TTCTTCAGGC CCCAGCCCTG 3600 GCAGGGGTCT GACCCCAGCA GGTAAGCAGAGAGTACTCCC TCCCCAGGAA GTGGAGGAGG 3660 GGACTCTGGG AATGGGGAAA TATGGTGCCCCATCCTGAAG CCAGCTGGTA CCTCCAGTTT 3720 GCACAGGGAC TTGTTGGGGG CTGAGGGCCCTGCCTACCCT TGGTGCTGTC ATAAAAGGGC 3780 AGGCGGGAGC GGGCTGAGAA ACAGCCTGTGCCTCCCAGAG ACTGACTCAG AGAGCCAGAG 3840 ACGTGTGTGT GTGTGTGTGT GTGTGTGTGTGTGTGTGTGT GTGTGTGAAA GACGGGGGTG 3900 GGGTATGTAT GCGTGTGTTG TGCACATGCTTGCCTGCACA GAGAGCATGA GTGTGTACAA 3960 GCTTAGCCCT GTGCCCTGTA GTGGGGCCAGCTGGGCAGAC AGCGAAATAA AAGGCAATAA 4020 GTTGAAA 4027 4027 base pairsnucleic acid single linear cDNA to mRNA rat skeletal muscle myoblast L6CDS 262..3243 by similarity to some other pattern 4 GAAAAATGAAGATCTATACC GACAGCAGAT CAGTGGCTGC CTGGGGCAAA GTTGGAGGGA 60 CATGTTATTTTGATTGTGAT GACATAATAC ATGCAAACAC GGCTAATCCT CTCAAAGCAT 120 ACACTTATACATGTGCAGCT TGGTATACAT AAATTATCCA TTACAAAACT ATGAGAAAGC 180 TATCACCACTATGAAGCACC ACTCACAGTA TGTGAATCTC CACCCCCCTT CCACTGCTGA 240 GACACAGAAATCCTAGACTG G ATG GAG AAC CCC TAC GTT GGG CGA GCG AGA 291 Met Glu Asn ProTyr Val Gly Arg Ala Arg 1 5 10 GCA GCA GCG GAG CGA GCA GCG GCA GAA GCCACG AAT TCA CTA TCG ATC 339 Ala Ala Ala Glu Arg Ala Ala Ala Glu Ala ThrAsn Ser Leu Ser Ile 15 20 25 CTG GTT CGG CCC ACC TCT GAA GGT TCC AGA ATCGAT AGT GAA TTC GTG 387 Leu Val Arg Pro Thr Ser Glu Gly Ser Arg Ile AspSer Glu Phe Val 30 35 40 GAG CTG GCA TGG ACA TCT CAT CCA GAG AGT GGG TGGGAA GAA GTG AGC 435 Glu Leu Ala Trp Thr Ser His Pro Glu Ser Gly Trp GluGlu Val Ser 45 50 55 GCC TAC GAT GAA GCC ATG AAT CCT ATC CGC ACG TAT CAGGTG TGT AAC 483 Ala Tyr Asp Glu Ala Met Asn Pro Ile Arg Thr Tyr Gln ValCys Asn 60 65 70 GTG CGC GAG TCC AGC CAG AAC AAC TGG CTG CGG ACC GGT TTCATC TGG 531 Val Arg Glu Ser Ser Gln Asn Asn Trp Leu Arg Thr Gly Phe IleTrp 75 80 85 90 CGG CGG GAA GTC CAG CGC GTC TAC GTG GAG CTG AAG TTT ACCGTG AGA 579 Arg Arg Glu Val Gln Arg Val Tyr Val Glu Leu Lys Phe Thr ValArg 95 100 105 GAT TGC AAC AGC ATC CCC AAC ATC CCT GGC TCC TGC AAG GAAACC TTC 627 Asp Cys Asn Ser Ile Pro Asn Ile Pro Gly Ser Cys Lys Glu ThrPhe 110 115 120 AAC CTT TTT TAC TAC GAG GCT GAT AGC GAT GTG GCG TCA GCCTCC TCT 675 Asn Leu Phe Tyr Tyr Glu Ala Asp Ser Asp Val Ala Ser Ala SerSer 125 130 135 CCC TTC TGG ATG GAG AAC CCC TAC GTG AAA GTG GAC ACC ATTGCG CCA 723 Pro Phe Trp Met Glu Asn Pro Tyr Val Lys Val Asp Thr Ile AlaPro 140 145 150 GAT GAG AGC TTC TCG CGG CTA GAC GCT GGG CGC GTT AAC ACCAAA GTG 771 Asp Glu Ser Phe Ser Arg Leu Asp Ala Gly Arg Val Asn Thr LysVal 155 160 165 170 CGC AGC TTC GGG CCG CTT TCC AAA GCC GGC TTC TAC TTGGCC TTC CAG 819 Arg Ser Phe Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu AlaPhe Gln 175 180 185 GAC CAG GGT GCC TGC ATG TCA CTC ATC TCT GTG CGC GCCTTC TAC AAG 867 Asp Gln Gly Ala Cys Met Ser Leu Ile Ser Val Arg Ala PheTyr Lys 190 195 200 AAG TGT GCA TCC ACC ACT GCA GGC TTC GCA CTC TTC CCCGAG ACC CTC 915 Lys Cys Ala Ser Thr Thr Ala Gly Phe Ala Leu Phe Pro GluThr Leu 205 210 215 ACG GGG GCT GAG CCC ACT TCG CTG GTC ATT GCC CCT GGCACC TGC ATC 963 Thr Gly Ala Glu Pro Thr Ser Leu Val Ile Ala Pro Gly ThrCys Ile 220 225 230 GCT AAC GCT GTG GAG GTG TCT GTA CCG CTC AAG CTC TACTGC AAT GGC 1011 Ala Asn Ala Val Glu Val Ser Val Pro Leu Lys Leu Tyr CysAsn Gly 235 240 245 250 GAC GGG GAG TGG ATG GTG CCC GTT GGT GCC TGC ACCTGC GCT ACT GGC 1059 Asp Gly Glu Trp Met Val Pro Val Gly Ala Cys Thr CysAla Thr Gly 255 260 265 CAT GAG CCA GCC GCC AAG GAG ACC CAG TGC CGC GCCTGT CCC CCT GGG 1107 His Glu Pro Ala Ala Lys Glu Thr Gln Cys Arg Ala CysPro Pro Gly 270 275 280 AGC TAC AAG GCA AAG CAA GGA GAG GGG CCC TGC CTCCCC TGT CCC CCC 1155 Ser Tyr Lys Ala Lys Gln Gly Glu Gly Pro Cys Leu ProCys Pro Pro 285 290 295 AAT AGC CGC ACC ACC TCG CCG GCT GCC AGC ATC TGCACC TGT CAC AAT 1203 Asn Ser Arg Thr Thr Ser Pro Ala Ala Ser Ile Cys ThrCys His Asn 300 305 310 AAT TTC TAC CGC GCA GAC TCA GAC ACA GCG GAC AGCGCC TGC ACC ACG 1251 Asn Phe Tyr Arg Ala Asp Ser Asp Thr Ala Asp Ser AlaCys Thr Thr 315 320 325 330 GTG CCG TCT CCC CCC CGG GGT GTG ATC TCC AATGTG AAT GAG ACC TCG 1299 Val Pro Ser Pro Pro Arg Gly Val Ile Ser Asn ValAsn Glu Thr Ser 335 340 345 CTG ATC CTC GAG TGG AGT GAG CCC CGG GAC CTTGGC GGA CGA GAT GAC 1347 Leu Ile Leu Glu Trp Ser Glu Pro Arg Asp Leu GlyGly Arg Asp Asp 350 355 360 CTC CTT TAT AAT GTT ATC TGT AAG AAG TGC CGTGGC AGC TCT GGG GCT 1395 Leu Leu Tyr Asn Val Ile Cys Lys Lys Cys Arg GlySer Ser Gly Ala 365 370 375 GGA GGT CCG GCG ACC TGT TCA CGC TGT GAT GACAAC GTG GAG TTC GAG 1443 Gly Gly Pro Ala Thr Cys Ser Arg Cys Asp Asp AsnVal Glu Phe Glu 380 385 390 CCC CGA CAG CTG GGC CTG ACC GAG CGC CGG GTCCAC ATC AGC CAC CTG 1491 Pro Arg Gln Leu Gly Leu Thr Glu Arg Arg Val HisIle Ser His Leu 395 400 405 410 TTG GCC CAC ACC CGC TAC ACC TTT GAG GTGCAG GCT GTC AAC GGC GTC 1539 Leu Ala His Thr Arg Tyr Thr Phe Glu Val GlnAla Val Asn Gly Val 415 420 425 TCT GGC AAA AGC CCT TTG CCG CCC CGC TATGCA GCT GTG AAT ATC ACC 1587 Ser Gly Lys Ser Pro Leu Pro Pro Arg Tyr AlaAla Val Asn Ile Thr 430 435 440 ACC AAC CAG GCC GCC CCA TCA GAA GTG CCTACG CTC CAC TTG CAC AGC 1635 Thr Asn Gln Ala Ala Pro Ser Glu Val Pro ThrLeu His Leu His Ser 445 450 455 AGT TCA GGG AGC AGC CTG ACC CTG TCC TGGGCA CCC CCG GAG CGG CCT 1683 Ser Ser Gly Ser Ser Leu Thr Leu Ser Trp AlaPro Pro Glu Arg Pro 460 465 470 AAC GGA GTC ATC TTG GAC TAT GAG ATG AAGTAC TTT GAG AAG AGT AAA 1731 Asn Gly Val Ile Leu Asp Tyr Glu Met Lys TyrPhe Glu Lys Ser Lys 475 480 485 490 GGC ATC GCC TCC ACT GTC ACC AGC CAGAAG AAC TCT GTA CAA CTG GAC 1779 Gly Ile Ala Ser Thr Val Thr Ser Gln LysAsn Ser Val Gln Leu Asp 495 500 505 GGA CTG CAG CCC GAC GCC CGC TAT GTAGTT CAG GTC CGG GCT CGC ACA 1827 Gly Leu Gln Pro Asp Ala Arg Tyr Val ValGln Val Arg Ala Arg Thr 510 515 520 GTA GCA GGT TAC GGA CAG TAT AGC CGCCCA GCT GAG TTT GAG ACC ACG 1875 Val Ala Gly Tyr Gly Gln Tyr Ser Arg ProAla Glu Phe Glu Thr Thr 525 530 535 AGT GAA AGA GGC TCA GGG GCC CAG CAGCTT CAA GAG CAG CTT CCC CTA 1923 Ser Glu Arg Gly Ser Gly Ala Gln Gln LeuGln Glu Gln Leu Pro Leu 540 545 550 ATT GTG GGA TCC ACC GTA GCT GGC TTTGTC TTC ATG GTG GTC GTC GTG 1971 Ile Val Gly Ser Thr Val Ala Gly Phe ValPhe Met Val Val Val Val 555 560 565 570 GTC ATT GCT CTT GTC TGC CTC AGGAAG CAG CGC CAG GGC CCT GAT GCA 2019 Val Ile Ala Leu Val Cys Leu Arg LysGln Arg Gln Gly Pro Asp Ala 575 580 585 GAA TAC ACG GAG AAG TTG CAG CAATAC GTT GCC CCC AGG ATG AAA GTT 2067 Glu Tyr Thr Glu Lys Leu Gln Gln TyrVal Ala Pro Arg Met Lys Val 590 595 600 TAC ATT GAC CCC TTT ACC TAC GAGGAT CCC AAT GAG GCC GTC CGA GAG 2115 Tyr Ile Asp Pro Phe Thr Tyr Glu AspPro Asn Glu Ala Val Arg Glu 605 610 615 TTC GCC AAG GAG ATC GAT GTG TCCTGC GTC AAG ATC GAG GAG GTG ATT 2163 Phe Ala Lys Glu Ile Asp Val Ser CysVal Lys Ile Glu Glu Val Ile 620 625 630 GGA GCT GGG GAG TTT GGG GAA GTGTGC CGG GGT CGG CTG AAA CTG CCC 2211 Gly Ala Gly Glu Phe Gly Glu Val CysArg Gly Arg Leu Lys Leu Pro 635 640 645 650 GGC CGC CGG GAG GTG TTC GTGGCC ATC AAG ACA CTG AAG GTG GGA TAC 2259 Gly Arg Arg Glu Val Phe Val AlaIle Lys Thr Leu Lys Val Gly Tyr 655 660 665 ACG GAG AGG CAG CGG CGG GACTTC CTG AGT GAG GCT TCC ATC ATG GGT 2307 Thr Glu Arg Gln Arg Arg Asp PheLeu Ser Glu Ala Ser Ile Met Gly 670 675 680 CAA TTT GAC CAT CCA AAT ATAATC CGT CTA GAG GGC GTG GTC ACC AAA 2355 Gln Phe Asp His Pro Asn Ile IleArg Leu Glu Gly Val Val Thr Lys 685 690 695 AGT CGT CCA GTC ATG ATC CTCACT GAG TTC ATG GAG AAC TGT GCC CTG 2403 Ser Arg Pro Val Met Ile Leu ThrGlu Phe Met Glu Asn Cys Ala Leu 700 705 710 GAC TCC TTC CTA CGG CTC AATGAC GGG CAG TTC ACA GTC ATC CAG CTT 2451 Asp Ser Phe Leu Arg Leu Asn AspGly Gln Phe Thr Val Ile Gln Leu 715 720 725 730 GTG GGC ATG TTG CGT GGCATT GCT GCC GGC ATG AAG TAC TTG TCT GAG 2499 Val Gly Met Leu Arg Gly IleAla Ala Gly Met Lys Tyr Leu Ser Glu 735 740 745 ATG AAC TAC GTG CAC CGTGAC CTC GCT GCC CGC AAC ATC CTT GTC AAC 2547 Met Asn Tyr Val His Arg AspLeu Ala Ala Arg Asn Ile Leu Val Asn 750 755 760 AGT AAC TTG GTC TGC AAAGTA TCT GAC TTT GGG CTC TCC CGC TTC CTG 2595 Ser Asn Leu Val Cys Lys ValSer Asp Phe Gly Leu Ser Arg Phe Leu 765 770 775 GAG GAC GAC CCC TCA GACCCC ACC TAC ACC AGC TCC CTG GGT GGG AAG 2643 Glu Asp Asp Pro Ser Asp ProThr Tyr Thr Ser Ser Leu Gly Gly Lys 780 785 790 ATC CCT ATC CGT TGG ACCGCC CCA GAG GCC ATA GAC TAT CGG AAG TTC 2691 Ile Pro Ile Arg Trp Thr AlaPro Glu Ala Ile Asp Tyr Arg Lys Phe 795 800 805 810 ACG TCT GCC AGC GATGTC TGG AGC TAC GGG ATC GTC ATG TGG GAG GTC 2739 Thr Ser Ala Ser Asp ValTrp Ser Tyr Gly Ile Val Met Trp Glu Val 815 820 825 ATG AGC TAC GGA GAGCGA CCA TAC TGG GAC ATG AGC AAC CAG GAT GTC 2787 Met Ser Tyr Gly Glu ArgPro Tyr Trp Asp Met Ser Asn Gln Asp Val 830 835 840 ATC AAT GCC GTA GAGCAA GAC TAT CGG TTA CCA CCC CCC ATG GAC TGC 2835 Ile Asn Ala Val Glu GlnAsp Tyr Arg Leu Pro Pro Pro Met Asp Cys 845 850 855 CCA GCG GCG CTG CACCAG CTC ATG CTG GAC TGT TGG GTG CGG GAC CGG 2883 Pro Ala Ala Leu His GlnLeu Met Leu Asp Cys Trp Val Arg Asp Arg 860 865 870 AAC CTC AGG CCC AAGTTC TCC CAA ATC GTC AAC ACG CTA GAC AAG CTT 2931 Asn Leu Arg Pro Lys PheSer Gln Ile Val Asn Thr Leu Asp Lys Leu 875 880 885 890 ATC CGC AAT GCTGCC AGC CTC AAG GTC ATC GCC AGT GCC CCA TCT GGC 2979 Ile Arg Asn Ala AlaSer Leu Lys Val Ile Ala Ser Ala Pro Ser Gly 895 900 905 ATG TCC CAG CCCCTC CTA GAC CGC ACG GTC CCA GAT TAT ACG ACC TTC 3027 Met Ser Gln Pro LeuLeu Asp Arg Thr Val Pro Asp Tyr Thr Thr Phe 910 915 920 ACG ACG GTG GGCGAC TGG CTA GAT GCC ATC AAG ATG GGG AGG TAT AAA 3075 Thr Thr Val Gly AspTrp Leu Asp Ala Ile Lys Met Gly Arg Tyr Lys 925 930 935 GAG AGC TTC GTCGGT GCG GGT TTT GCC TCC TTT GAC CTG GTG GCC CAG 3123 Glu Ser Phe Val GlyAla Gly Phe Ala Ser Phe Asp Leu Val Ala Gln 940 945 950 ATG ACT GCA GAAGAT CTG CTA AGG ATC GGG GTC ACT TTG GCC GGC CAC 3171 Met Thr Ala Glu AspLeu Leu Arg Ile Gly Val Thr Leu Ala Gly His 955 960 965 970 CAG AAG AAGATC CTC AGC AGT ATC CAG GAC ATG CGG CTG CAG ATG AAC 3219 Gln Lys Lys IleLeu Ser Ser Ile Gln Asp Met Arg Leu Gln Met Asn 975 980 985 CAG ACA CTGCCC GTG CAG GTC TGACGCTCAG CTCCAGCGAG GGGCGTGGCC 3270 Gln Thr Leu ProVal Gln Val 990 CCCCGGGACT GCACAAGGAT TCTGACCAGC CAGCTGGACT TTTGGATACCTGGCCTTTGG 3330 CTGTGGCCCA GAAGACAGAA GTTCGGGGGA GAACCCTAGC TGTGACTTCTCCAAGCCTGT 3390 GCTCCCTCCC AGGAAGTGTG CCCCAAACCT CTTCATATTG AAGATGGATTAGAAGAGGGG 3450 GTGATATCCC CTCCCCAGAT GCCTCAGGGC CCAGGCCTGC CTGCTCTCCAGTCGGGGATC 3510 TTCACAACTC AGATTTGGTT GTGCTTCAGT AGTGGAGGTC CTGGTAGGGTCGGGTGGGGA 3570 TAAGCCTGGG TTCTTCAGGC CCCAGCCCTG GCAGGGGTCT GACCCCAGCAGGTAAGCAGA 3630 GAGTACTCCC TCCCCAGGAA GTGGAGGAGG GGACTCTGGG AATGGGGAAATATGGTGCCC 3690 CATCCTGAAG CCAGCTGGTA CCTCCAGTTT GCACAGGGAC TTGTTGGGGGCTGAGGGCCC 3750 TGCCTACCCT TGGTGCTGTC ATAAAAGGGC AGGCGGGAGC GGGCTGAGAAACAGCCTGTG 3810 CCTCCCAGAG ACTGACTCAG AGAGCCAGAG ACGTGTGTGT GTGTGTGTGTGTGTGTGTGT 3870 GTGTGTGTGT GTGTGTGAAA GACGGGGGTG GGGTATGTAT GCGTGTGTTGTGCACATGCT 3930 TGCCTGCACA GAGAGCATGA GTGTGTACAA GCTTAGCCCT GTGCCCTGTAGTGGGGCCAG 3990 CTGGGCAGAC AGCGAAATAA AAGGCAATAA GTTGAAA 4027 11 aminoacids amino acid linear protein rat skeletal muscle myoblast L6 5 ValIle Gly Ala Gly Glu Phe Gly Glu Val Cys 1 5 10 10 amino acids amino acidlinear protein rat skeletal muscle myoblast L6 6 Asn Ile Leu Val Asn SerAsn Leu Val Cys 1 5 10 5 amino acids amino acid linear protein ratskeletal muscle myoblast L6 7 Val Glu Gln Asp Tyr 1 5 28 base pairsnucleic acid single linear DNA (synthetic) 8 GTAATACGAC TCACTATAGGGGAGAGCT 28 28 base pairs nucleic acid single linear DNA (synthetic) 9CTCCCCTATA GTGAGTCGTA TTACTGCA 28 32 base pairs nucleic acid singlelinear DNA (synthetic) 10 CTAGTCTATA GTGTCACCTA AATCGTGGGT AC 32 23 basepairs nucleic acid single linear DNA (synthetic) 11 CACGATTTAGGTGACACTAT AGA 23 44 base pairs nucleic acid single linear DNA(synthetic) 12 AATATAGTCG ACCACCATGG AGAACCCCTA CGTTGGGCGA GCGA 44 37base pairs nucleic acid single linear DNA (synthetic) 13 CGGCGGACTAGTTCAGACCT GCACGGGCAG TGTCTGG 37 55 base pairs nucleic acid singlelinear DNA (synthetic) 14 GCCGCCACTA GTTCAGTGGT GGTGGTGGTG GTGGACCTGCACGGGCAGTG TCTGG 55 44 base pairs nucleic acid single linear DNA(synthetic) 15 CGGCGGACTA GTTCATGAGC CTCTTTCACT CGTGGTCTCA AACT 44 62base pairs nucleic acid single linear DNA (synthetic) 16 GCCGCCACTAGTTCAGTGGT GGTGGTGGTG GTGTGAGCCT CTTTCACTCG TGGTCTCAAA 60 CT 62

What is claimed is:
 1. A composition for the treatment of diabetescomprising a therapeutically effective dose of a compound which inducesphosphorylation of a tyrosine residue of protein p140, having the aminoacid sequence shown in SEQ ID NO: 1 as an active ingredient.